Inhibitory anti-factor xii/xiia monoclonal antibodies and their uses

ABSTRACT

The invention relates to inhibitory anti-factor XII/FXIIa antibodies and methods of their use.

The invention relates to inhibitory anti-factor XII/FXIIa antibodies andmethods of their use.

Factor XII (Hageman Factor) is a serum glycoprotein with a molecularweight of about 80 kDa. Besides an autoactivation by exposure tonegatively charged surfaces, factor XII is additionally activated bykallikrein by proteolytic cleavage to form alpha-factor XIIa, which isthen further converted, for example by trypsin, into beta-factor XIIa(FXIIa-β). Alpha-factor XIIa is composed of the N-terminal heavy chainof about 50 kDa, which contains the contact binding domain, and theC-terminal light chain of about 28 kDa, which contains the catalyticcenter. The heavy and light chains are connected by a disulfide bond.FXIIa-β is an active form of FXII of about 30 kDa, consisting of thecomplete light chain and a 2000 Da fragment of the heavy chain linked bya disulfide bond.

Vessel wall injury triggers sudden adhesion and aggregation of bloodplatelets, followed by the activation of the plasma coagulation systemand the formation of fibrin-containing thrombi, which occlude the siteof injury. These events are crucial to limit post-traumatic blood lossbut may also occlude diseased vessels leading to ischemia and infarctionof vital organs. In the waterfall model, blood coagulation proceeds by aseries of reactions involving the activation of zymogens by limitedproteolysis culminating in generation of thrombin, which converts plasmafibrinogen to fibrin and activates platelets. In turn, collagen- orfibrin-adherent platelets facilitate thrombin generation by severalorders of magnitude via exposing procoagulant phospholipids (mainlyphosphatidyl serine) on their outer surface, which propagates assemblyand activation of coagulation protease complexes and by directinteraction between platelet receptors and coagulation factors.

Two converging pathways for coagulation exist that are triggered byeither extrinsic (vessel wall) or intrinsic (blood-borne) components ofthe vascular system. The “extrinsic” pathway is initiated by the complexof the plasma factor VII (FVII) with the integral membrane proteintissue factor (TF), an essential coagulation cofactor that is absent onthe luminal surface but strongly expressed in subendothelial layers ofthe vessel and which is accessible or liberated via tissue injury. TFexpressed in circulating microvesicles might also contribute to thrombuspropagation by sustaining thrombin generation on the surface ofactivated platelets. The “intrinsic” or contact activation pathway isinitiated when factor XII (FXII, Hageman factor) comes into contact withnegatively charged surfaces in a reaction involving high molecularweight kininogen and plasma kallikrein. FXII can be activated bymacromolecular constituents of the subendothelial matrix such asglycosaminoglycans and collagens, sulfatides, nucleotides,polyphosphates and other soluble polyanions or non-physiologicalmaterial such as glass or polymers. One of the most potent contactactivators is kaolin and this reaction serves as the mechanistic basisfor the major clinical clotting test, the activated partialthromboplastin time (aPTT), which measures the coagulation capacity viathe “intrinsic” pathway. In reactions propagated by platelets, activatedFXII then activates FXI to FXIa and subsequently FXIa activates factorIX. The complex of FVIIIa, which FVIIIa has been previously activated bytraces of FXa and/or thrombin, and FIXa (the tenase complex)subsequently activates FX.

Despite its high potency to induce blood clotting in vitro, the (patho-)physiological significance of the FXII-triggered intrinsic coagulationpathway is questioned by the fact that hereditary deficiencies of FXIIas well as of high molecular weight kininogen and plasma kallikrein arenot associated with bleeding complications. Together with theobservation that humans and mice lacking extrinsic pathway constituentssuch as TF and FVII suffer from severe bleeding this has led to thecurrent hypothesis that the cessation of bleeding in vivo requiresexclusively the extrinsic cascade (Mackman, N. 2004. Role of tissuefactor in hemostasis, thrombosis, and vascular development.Arterioscler. Thromb. Vasc. Biol. 24, 101 5-1 022).

In pathological conditions, the coagulation cascade may be activatedinappropriately which then results in the formation of haemostatic plugsinside the blood vessels. Thereby, vessels can be occluded and the bloodsupply to distal organs limited. This process is known as thrombosis,and, if the thrombus embolizes, as thromboembolism which is associatedwith high mortality. In addition, the use of prosthetic devices, whichcome into contact with blood, is severely limited because of activationof the intrinsic coagulation cascade. Suitable coating of the prostheticsurface may avoid said problem in some cases but may compromise itsfunction in others. Examples of such prosthetic devices arehemodialysers, cardiopulmonary bypass circuits, heart valves, vascularstents and in-dwelling catheters. In cases where such devices are used,anticoagulants, such as heparin, are administered to prevent fibrinformation on the surface. However, some patients are intolerant ofheparin, which can cause heparin-induced thrombocytopenia (HIT)resulting in platelet aggregation and life-threatening thrombosis.Furthermore, an inherent disadvantage of all anticoagulants used inclinics is an increased risk of serious bleeding events. Therefore, astrong need for new types of anticoagulants exist, which are notassociated with such complications and that can be used in affectedpatients or as superior prophylaxis/therapy concept preventingthrombosis without increased bleeding risks.

For more than five decades it has been known that deficiency ofcoagulation factor XII is not associated with increased spontaneous orinjury-related bleeding complications (Ratnoff O D & Colopy J E 1955. Afamilial hemorrhagic trait associated with a deficiency of aclot-promoting fraction of plasma. J Clin Invest 34:602-613). Indeed,although readily detected by a pathological value measured in the aPTT(a clinical clotting test that addresses the intrinsic pathway ofcoagulation) humans that are deficient in FXII do not suffer fromabnormal bleeding even during major surgical procedures (Colman R W.Hemostasis and Thrombosis. Basic principles & clinical practice (eds.Colman R W, Hirsch J, Mader V J, Clowes A W, & George J) 103-122(Lippincott Williams & Wilkins, Philadelphia, 2001)). In contrast,deficiency of FXII had been associated with increased risk of venousthrombosis (Kuhli C et al. 2004. Factor XII deficiency: a thrombophilicrisk factor for retinal vein occlusion. Am. J. Ophthalmol. 137:459-464;Halbmayer W M et al. 1993. Factor XII (Hageman factor) deficiency: arisk factor for development of thromboembolism. Incidence of FXIIdeficiency in patients after recurrent venous or arterialthromboembolism and myocardial infarction. Wien. Med. Wochenschr.143:43-50). Studies and case reports supporting this idea refer to theindex case for FXII deficiency, Mr. John Hageman, who died of pulmonaryembolism. The hypothesis that FXII deficiency is associated with anincreased prothrombotic risk is challenged by a recent reevaluation ofseveral case reports the original reports of which linked FXIIdeficiency with thrombosis (Girolami A et al. 2004. The occasionalvenous thromboses seen in patients with severe (homozygous) FXIIdeficiency are probably due to associated risk factors: A study ofprevalence in 21 patients and review of the literature. J. Thromb.Thrombolysis 17:139-143). In most cases the authors identifiedconcomitant congenital or acquired prothrombotic risk factors incombination with factor FXII deficiency that could be responsible forthe thrombotic event independently of FXII. The largest epidemiologicalstudies using well characterized patients (Koster T et al. 1994. JohnHageman's factor and deep-vein thrombosis: Leiden thrombophilia Study.Br. J. Haematol. 87:422-424) and FXII-deficient families (Zeerleder S etal. 1999. Reevaluation of the incidence of thromboembolic complicationsin congenital factor XII deficiency—a study on 73 subjects from 14 Swissfamilies. Thromb. Haemost. 82:1240-1246) indicated that there is nocorrelation of FXII deficiency and any pro- or antithrombotic risk.Surprisingly and in contrast to common believe of those skilled in theart it has been discovered that the factor XII-driven intrinsiccoagulation pathway is involved in arterial thrombus formation in vivobut is not necessary for normal tissue-specific hemostasis (Renne T etal. 2005. Defective thrombus formation in mice lacking factor XII. J.Exp. Med. 202:271-281; Kleinschnitz C et al. 2006. Targeting coagulationfactor XII provides protection from pathological thrombosis in cerebralischemia without interfering with hemostasis. J. Exp. Med. 203, 513-518;WO2006066878). Unexpectedly, these results place factor XII in a centralposition in the process of pathological thrombus formation. Hencesubstances capable of interfering and blocking FXII activation or FXIIactivity may be suited to block pathogenic arterial thrombus formationand the clinical consequences thereof.

In WO2006066878 the use of antibodies against FXII/FXIIa or the use ofinhibitors of FXII/FXIIa is proposed. As potential inhibitorsantithrombin III (AT III), angiotensin converting enzyme inhibitor, C1inhibitor, aprotinin, alpha-I protease inhibitor, antipain([(S)-I-Carboxy-2-Phenylethyl]-Carbamoyl-L-Arg-L-Val-Arginal),Z-Pro-Proaldehyde-dimethyl acetate, DX88 (Dyax Inc., 300 TechnologySquare, Cambridge, Mass. 02139, USA; cited in: Williams A and Baird L G.2003. DX-88 and HAE: a developmental perspective. Transfus ApheresisSci. 29:255-258), leupeptin, inhibitors of prolyl oligopeptidase such asFmoc-Ala-Pyr-CN, corn-trypsin inhibitor, mutants of the bovinepancreatic trypsin inhibitor, ecotin, yellowfin sole anticoagulantprotein, Cucurbita maxima trypsin inhibitor-V including Curcurbitamaxima isoinhibitors and Hamadarin (as disclosed by lsawa H et al. 2002.A mosquito salivary protein inhibits activation of the plasma contactsystem by binding to factor XII and high molecular weight kininogen. J.Biol. Chem. 277:27651-27658) have been proposed.

An ideal inhibitor of FXII/FXIIa as a therapeutic agent—while exhibitinga high inhibitory activity towards FXII/FXIIa—will not increase the riskof bleeding, be non-immunogenic and have to be administered as sparinglyas possible—ideally only once. Small molecule inhibitors likeZ-Pro-Pro-aldehyde-dimethyl acetate will have only a very shorthalf-life after administration, thus requiring multiple injections, orwould have to be developed into orally available slow release forms andthen also be given constantly over a long period. Human plasma proteinslike C1 inhibitor would at first sight fulfill all requirements, havinga relatively high inhibitory activity towards FXII/FXIIa while notincreasing the risk of bleeding, being non-immunogenic as a humanprotein and also having a considerably long plasma half-life. It was nowsurprisingly found that in an in vivo model of thrombosis C1 inhibitoras a prime candidate of a human FXII/FXIIa inhibitor could not be usedsuccessfully to prevent occlusion. Another proposed FXII/FXIIa inhibitorfrom human plasma namely AT III inhibitor would at least not fulfill thesecond requirement as the bleeding risk would increase (Warren B L etal. 2001. Caring for the critically ill patient. High-dose antithrombinIII in severe sepsis: a randomized controlled trial. JAMA286:1869-1878).

In WO2008098720A1 the use of Kazal-type serine protease inhibitorInfestin or domains thereof or modified Kazal-type serine proteaseinhibitors based on Infestin homologs as inhibitors of FXII/FXIIa isproposed. Selected from this subset, recombinant Infestin-4 fused tohuman albumin for prolongation of half-life (rHA-Infestin-4) wasdeveloped demonstrating high inhibitory activity towards FXII/FXIIa.Moreover this substance demonstrated antithrombotic efficacy withoutimpairing (physiologic) hemostasis while demonstrating a usefulhalf-life after fusion to human albumin (Hagedorn et al. 2010. FactorXIIa Inhibitor Recombinant Human Albumin Infestin-4 Abolishes OcclusiveArterial Thrombus Formation Without Affecting Bleeding. Circulation.121:1510-1517). However, although immunogenicity was reduced duringdevelopment, there is still the risk of immunogenic responses in man.Furthermore, an even longer half-life would have additional beneficialeffects. Hence, it is apparent that there still exists a need for animproved medication for the treatment and/or prophylaxis of thrombosisand similar disorders. Therefore, it is an object of the presentinvention to satisfy such a need. A candidate for such an improvedmedication is an improved anti-FXII/FXIIa antibody with inhibitoryactivity.

Antibodies to Factor XII have been disclosed. Pixley et al (J Biol Chem(1987) 262, 10140-10145) disclosed monoclonal antibody B7C9 to humanFactor XII. This antibody blocked surface-mediated coagulant activity,but not amidolytic activity of Factor XIIa. Small et al (Blood (1985),65, 202-210) disclosed a monoclonal antibody to human Factor XII, whichprevented activation of Factor XII, but not the coagulant or theamidolytic activity of activated FXII (FXIIa). Nuijens et al (J. Biol.Chem. (1989) 264, 12941-12949) disclosed monoclonal antibodies F1 andF3, which inhibited coagulation activity but not amidolytic activity ofFXII. WO8911865 provides monoclonal antibodies produced against thelight chain of FXII (B6F5, C6B7, D2E10). These antibodies inhibit thecoagulation activity, but only show partial inhibition of the amidolyticactivity of FXIIa. WO9008835 describes the production of monoclonalantibody that selectively binds FXIIa-3 over FXII, and the developmentof an immunoassay that specifically detects FXIIa-3 in blood. Fromexample 7 in WO9008835, it is clear that the antibody does not inhibitamidolytic activity of FXIIa. WO9117258 describes the treatment ofsepsis with an anti-FXII antibody OT-2, which binds to native FXII inplasma, and inhibits activation of the contact system in plasma, as wellas amidolytic activity of FXIIa.

An objective of the present invention was the development of an improvedantibody which—while exhibiting a high inhibitory activity towardsFXIIa—will not increase the risk of bleeding, be non-immunogenic andhave a long half-life. Since FXII has a multidomain structure includingfibronectin type and EGF-like domains (reviewed by Stavrou and Schmaier(2010) Thromb. Res., 125:210-215), it was believed that FXII should haveadditional important physiologic functions in addition to its role asFXIIa, i.e. as the enzyme following activation. New studies havedemonstrated now that FXII contributes to cell proliferation and growthleading to angiogenesis (reviewed by Schmaier and LaRusch (2010) Thromb.Haemost., 104:915-918). Therefore, in order not to interfere with these(and maybe other so far unknown) functions of FXII, it is preferable fora therapeutic antibody against FXII/FXIIa to have a clear higheraffinity towards FXIIa, for example towards FXIIa-β, compared to FXII.

SUMMARY OF THE INVENTION

One aspect of the invention is therefore an anti-Factor XII/FXIIamonoclonal antibody or antigen-binding fragment thereof that has a morethan 2 fold higher binding affinity to human Factor XIIa-beta than tohuman Factor XII and that is capable of inhibiting the amidolyticactivity of human Factor XIIa. Another aspect of the invention is ananti-Factor XII/XIIa monoclonal antibody or antigen-binding fragmentthereof, that inhibits human Factor XIIa-alpha by more than 50% whenused at a molar ratio of FXIIa-alpha to antibody of 1:0.2.

Preferably, the antibody or antigen-binding fragment thereof has one ormore of the following features:

-   -   it binds murine FXII/FXIIa;    -   the level of binding of the antibody to a polypeptide comprising        SEQ ID NO: 2 or relevant fragment thereof in which (a) the        asparagine residue at position 398 of SEQ ID NO: 2 is        substituted for lysine; or (b) the isoleucine residue at        position 438 of SEQ ID NO: 2 is substituted for alanine, is        lower than the level of binding of the protein to the        corresponding polypeptide comprising SEQ ID NO: 2 or relevant        fragment thereof without said substitution;    -   It comprises a heavy chain variable (vH) region which is more        than 85% identical to the sequence of SEQ ID NO: 4;    -   it comprises a light chain variable (vL) region which is more        than 85% identical to the sequence of SEQ ID NO: 5;    -   it comprises heavy chain CDR1 at least 80% identical to the        sequence of SEQ ID NO: 6, and/or heavy chain CDR2 at least 60%        identical with SEQ ID NO: 7, and/or heavy chain CDR3 at least        80% identical to the sequence of SEQ ID NO: 9;    -   it comprises light chain CDR1 at least 50% identical with SEQ ID        NO: 11, and/or light chain CDR2 of SEQ ID NO: 12, and/or light        chain CDR3 with the sequence A-X₁-W-X₂-X₃-X₄-X₅-R-X₆-X₇ wherein        X₁ can be A or S, X₅ can be L or V, the other X_(n)s can be any        amino acid (SEQ ID NO: 14).    -   it binds human Factor XIIa-beta with a K_(D) of better than        10⁻⁸M.    -   it competes with Infestin, in particular with infestin-4, for        binding to human Factor XIIa-beta.    -   it is a human IgG or variant thereof, preferably human IgG4 or        variant thereof.

Another aspect of the invention is a nucleic acid encoding the antibody,or antigen-binding fragment thereof, of the invention.

Yet another aspect of the invention is a vector comprising the nucleicacid encoding the antibody, or antigen-binding fragment thereof, of theinvention, operably linked to a suitable promoter sequence.

A further aspect of the invention is a cell line or yeast cellcomprising the vector of the invention.

Another aspect of the invention is a method of producing the antibody orantigen binding fragment thereof of the invention, comprising culturingthe cell line or yeast cell of the invention under appropriateconditions to express the antibody or antigen binding fragment thereof,and purifying the antibody or antigen binding fragment thereof from theculture supernatant.

Yet another aspect of the invention is the antibody or antigen bindingfragment thereof for medical use.

A further aspect of the invention is the antibody or antigen bindingfragment thereof for use in preventing and/or treating a disorderselected from the group consisting of venous, arterial or capillarythrombus formation, thrombus formation in the heart, thrombus formationduring and/or after contacting blood of a human or animal subject withartificial surfaces, thromboembolism, by preventing the formation and/orthe stabilization of thrombi and thereby three-dimensional intraluminalthrombus growth, or by preventing and/or treating intraluminal thrombi;interstitial lung disease, inflammation, a neurological inflammatorydisease, complement activation, fibrinolysis, angiogenesis and diseasesrelated to FXII/FXIIa-induced kinin formation or FXII/FXIIa-mediatedcomplement activation. Yet another aspect of the invention is theantibody or antigen-binding fragment thereof for use in the treatment ofintraluminal thrombi in a human or animal subject related to a disorderselected from the group consisting of venous, arterial or capillarythrombus formation, thrombus formation in the heart, thrombus formationduring and/or after contacting blood of a human or animal subject withartificial surfaces, thromboembolism; interstitial lung disease,inflammation, a neurological inflammatory disease, complementactivation, fibrinolysis, angiogenesis and diseases related toFXII/FXIIa-induced kinin formation or FXII/FXIIa-mediated complementactivation. Preferably, the venous or arterial thrombus formation isstroke, myocardial infarction, deep vein thrombosis, portal veinthrombosis, renal vein thrombosis, jugular vein thrombosis, cerebralvenous sinus thrombosis, Budd-Chiari syndrome or Paget-Schroetterdisease. Preferably, the diseases related to FXII/FXIIa-induced kininformation are selected from the group hereditary angioedema, bacterialinfections of the lung, trypanosoma infections, hypotensive shock,pancreatitis, chagas disease, articular gout, arthritis, disseminatedintravascular coagulation (DIC) and sepsis.

Preferably the interstitial lung disease is fibroproliferative and/oridiopathic pulmonary fibrosis.

Preferably, the thrombus formation occurs during and/or after contactingblood of a human or animal subject with artificial surfaces duringand/or after a medical procedure performed on said human or animalsubject and said antibody or antigen-binding fragment thereof isadministered before and/or during and/or after said medical procedure,and further

-   -   (i) the artificial surface is exposed to at least 80% of the        blood volume of the subject and the artificial surface is at        least 0.2 m² or    -   (ii) the artificial surface is a container for collection of        blood outside the body of the subject or    -   (iii) the artificial surface is a stent, valve, intraluminal        catheter, or a system for internal assisted pumping of blood.

Yet a further aspect of the invention is a medical device coated withthe antibody or antigen-binding fragment thereof of the invention,wherein the device is a cardiopulmonary bypass machine, anextracorporeal membrane oxygenation system for oxygenation of blood, adevice for assisted pumping of blood, a blood dialysis device, a devicefor the extracorporeal filtration of blood, a repository for use in thecollection of blood, an intraluminal catheter, a stent, an artificialheart valve, and/or accessories for any one of said devices includingtubing, cannulae, centrifugal pump, valve, port, and/or diverter.

Another aspect of the invention is the antibody or antigen-bindingfragment thereof for use for administration in a patient receiving amedical procedure, wherein the medical procedure comprises contact withat least one of:

-   -   (a) heart,    -   (b) at least one blood vessel chosen from: the aorta, the aortic        arch, a carotid artery, a coronary artery, brachiocephalic        artery, vertebrobasilar circulation, intracranial arteries,        renal artery, a hepatic artery, a mesenteric artery, and/or a        blood vessel of the arterial system cranial to the heart,    -   (c) a venous blood vessel if the patient has a known septal        defect;        and wherein the medical procedure comprises release of at least        one embolus in at least one of said blood vessels in the body        that could result in ischemia in at least one target organ and        administration of the antibody or antigen binding fragment        thereof before, during and/or after the medical procedure.

Another aspect of the invention is the antibody or antigen bindingfragment thereof for use in the prevention or treatment of a conditionassociated with increased vascular permeability, in particular increasedretinal vascular permeability, including progressive retinopathy,sight-threatening complication of retinopathy, macular edema,non-proliferative retinopathy, proliferative retinopathy, retinal edema,diabetic retinopathy, hypertensive retinopathy, and retinal trauma.

Another aspect of the invention is a pharmaceutical compositioncomprising the antibody or antigen binding fragment thereof of theinvention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Anti-FXIIa phage competition ELISA using the FXIIa amidolyticinhibitor infestin4. The concentrations of the competitor (rHA-Inf4) areshown on the X-axis. Fixed concentrations of phage-expressed Fabantibody or infestin4 (pTaclnf4) used in the assay were determined usinga phage titration ELISA.

FIG. 2: Concentration-dependent inhibition of amidolytic activity ofhuman FXIIa by monoclonal antibody 3F7 as a fully human IgG4. Theanti-human GCSF receptor monoclonal antibody C1.2 (fully human IgG4) wasused as a negative control and rHA-Infestin as a positive control forthe assay.

FIG. 3: 3F7 heavy chain stop templates used for affinity maturation. CDRregions are shaded grey and amino acid positions in each library thatwere randomised are designated as “x”.

FIG. 4: 3F7 light chain stop templates used for affinity maturation. CDRregions are shaded grey and amino acid positions in each library thatwere randomised are designated as “x”.

FIG. 5: Concentration-dependent inhibition of amidolytic activity ofhuman FXIIa by monoclonal antibodies 3F7 and OT-2.

FIG. 6: A: Alignment of the catalytic domains of FXII of mouse, rat andhuman, and identification of the residues that form the catalytic triad(*) and the mutations introduced (!) to identify the potential epitopeof antibody 3F7. B: Western Blot showing the binding of 3F7 to thevarious mutants.

FIG. 7: Occlusion rate in FeCl₃-induced thrombosis following treatmentwith MAb 3F7 (n=5-25/group)

FIG. 8: Effect of MAb 3F7 on aPTT (n=5-25/group; mean±SD)

FIG. 9: Effect of MAb 3F7 on PT (n=5-25/group; mean±SD)

FIG. 10: Effect of MAb 3F7 on FXIIa-activity (n=5-25/group; mean±SD)

FIG. 11: Effect of MAb 3F7 on time to hemostasis. Data are presented asmean values (+SD). Statistics: p>0.05 (Kruskal-Wallis test). N=10/group.

FIG. 12: Effect of MAb 3F7 on total blood loss. Data are presented asmean values (+SD). Statistics: p>0.05 (Kruskal-Wallis test). N=10/group.

FIG. 13: Effect of MAb 3F7 on time to hemostasis. Horizontal linesrepresent median values. Statistics: p>0.05 (Kruskal-Wallis test).N=10/group.

FIG. 14: Effect of MAb 3F7 on total blood loss. Horizontal linesrepresent median values. Statistics: p>0.05 (Kruskal-Wallis test).N=10/group.

FIG. 15: Comparison of aPTT of OT-2, MAb 3F7 and affinity-maturedversions of MAb 3F7

FIG. 16: Comparison of inhibition of human Factor XIIa-alpha bydifferent antibodies

LIST OF SEQUENCES

SEQ ID NO: 1: Human FXII sequenceSEQ ID NO: 2: Mouse FXII sequenceSEQ ID NO: 3: Rat FXII sequenceSEQ ID NO: 4: 3F7 vH sequenceSEQ ID NO: 5: 3F7 vL sequenceSEQ ID NO: 6: 3F7 heavy chain CDR1 (HCDR1)SEQ ID NO: 7: 3F7 heavy chain CDR2 (HCDR2)SEQ ID NO: 8: 3F7 heavy chain CDR2 with variationSEQ ID NO: 9: 3F7 heavy chain CDR3 (HCDR3)SEQ ID NO: 10: 3F7 heavy chain CDR3 with variationSEQ ID NO: 11: 3F7 light chain CDR1 (LCDR1)SEQ ID NO: 12: 3F7 light chain CDR2 (LCDR2)SEQ ID NO: 13: 3F7 light chain CDR3 (LCDR3)SEQ ID NO: 14: 3F7 light chain CDR3 with variationSEQ ID NO: 15: 3F7 heavy chain stop template H1SEQ ID NO: 16: Oligonucleotide mutagenic trimer mix 3F7 H1SEQ ID NO: 17: 3F7 heavy chain stop template H2SEQ ID NO: 18: Oligonucleotide mutagenic trimer mix 3F7 H2SEQ ID NO: 19: 3F7 heavy chain stop template H3.1SEQ ID NO: 20: Oligonucleotide mutagenic trimer mix 3F7 H3.1SEQ ID NO: 21: 3F7 heavy chain stop template H3.2SEQ ID NO: 22: Oligonucleotide mutagenic trimer mix 3F7 H3.2SEQ ID NO: 23: 3F7 light chain stop template L1SEQ ID NO: 24: Oligonucleotide mutagenic trimer mix 3F7 L1SEQ ID NO: 25: 3F7 light chain stop template L3.1SEQ ID NO: 26: Oligonucleotide mutagenic trimer mix 3F7 L3.1SEQ ID NO: 27: 3F7 light chain stop template L3.2SEQ ID NO: 28: Oligonucleotide mutagenic trimer mix 3F7 L3.2SEQ ID NO: 29: VR119 heavy chain CDR2SEQ ID NO: 30: VR112 heavy chain CDR2SEQ ID NO: 31: VR115 heavy chain CDR2SEQ ID NO: 32: VR110 heavy chain CDR2SEQ ID NO: 33: VR107 heavy chain CDR2SEQ ID NO: 34: VR108 heavy chain CDR2SEQ ID NO: 35: VR103 heavy chain CDR2SEQ ID NO: 36: VR101 heavy chain CDR2SEQ ID NO: 37: VR109 heavy chain CDR2SEQ ID NO: 38: VR99 heavy chain CDR2SEQ ID NO: 39: VR149 heavy chain CDR3SEQ ID NO: 40: VR167 heavy chain CDR3SEQ ID NO: 41: VR148 heavy chain CDR3SEQ ID NO: 42: VR159 heavy chain CDR3SEQ ID NO: 43: VR160 heavy chain CDR3SEQ ID NO: 44: VR24 light chain CDR1SEQ ID NO: 45: VRO6 light chain CDR1SEQ ID NO: 46: VR16 light chain CDR1SEQ ID NO: 47: VRO5 light chain CDR1SEQ ID NO: 48: VR12 light chain CDR1SEQ ID NO: 49: VR10 light chain CDR1SEQ ID NO: 50: VR14 light chain CDR1SEQ ID NO: 51: VR17 light chain CDR1SEQ ID NO: 52: VR31 light chain CDR3SEQ ID NO: 53: VR29 light chain CDR3SEQ ID NO: 54: VR27 light chain CDR3SEQ ID NO: 55: VR39 light chain CDR3SEQ ID NO: 56: VR46 light chain CDR3SEQ ID NO: 57: VR41 light chain CDR3SEQ ID NO: 58: VR38 light chain CDR3SEQ ID NO: 59: VR58 light chain CDR3SEQ ID NO: 60: VR62 light chain CDR3SEQ ID NO: 61: VR53 light chain CDR3SEQ ID NO: 62: VR52 light chain CDR3SEQ ID NO: 63: VR63 light chain CDR3SEQ ID NO: 64: Sequencing primer CH1 RevSEQ ID NO: 65: Sequencing primer pLacPCRfwSEQ ID NO: 66: Sequencing primer wt GIII stump revSEQ ID NO: 67: Sequencing primer KpaCLfwdSEQ ID NO: 68: Sequencing primer LdaCLfwdSEQ ID NO: 69: Sequencing primer PUCrevSEQ ID NO: 70: Sequencing primer 3254SEQ ID NO: 71: Sequencing primer Seq CL lambdaSEQ ID NO: 72: Sequencing primer Seq CH1SEQ ID NO: 73: vH sequence of VR115SEQ ID NO: 74: vH sequence of VR112SEQ ID NO: 75: vL sequence of VR24SEQ ID NO: 76: vH sequence of VR110SEQ ID NO: 77: vH sequence of VR119

DETAILED DESCRIPTION OF THE INVENTION

An objective of the present invention was the development of an improvedantibody which—while exhibiting a high inhibitory activity towardsFXIIa—will not increase the risk of bleeding, be non-immunogenic andhave a long half-life.

One aspect of the invention is therefore an anti-Factor XII/FXIIamonoclonal antibody or antigen-binding fragment thereof that has a morethan 2 fold higher binding affinity to human Factor XIIa, preferably tohuman Factor XIIa-beta, than to human Factor XII and that is capable ofcompletely inhibiting the amidolytic activity of human Factor XIIa.

Another aspect of the invention is an antibody or antigen bindingfragment thereof that has a more than 2 fold higher binding affinity tohuman Factor XIIa, preferably to human Factor XIIa-beta, than to humanFactor XII and that is capable of completely inhibiting the amidolyticactivity of human Factor XIIa and that competes with an antibodycomprising the sequences of SEQ ID NOs: 4 and 75 expressed as IgG4 forthe binding to FXII/FXIIa.

Preferably the antibody or antigen binding fragment thereof has morethan 3 fold, more preferably more than 4 fold, even more preferably morethan 5 fold, more than 6 fold, more than 8 fold, more than 10 fold, morethan 12 fold, more than 14 fold, more than 16 fold, most preferably morethan 18 fold higher binding affinity to human Factor XIIa, preferably tohuman Factor XIIa-beta, than to human Factor XII.

Preferably, the antibody or antigen-binding fragment thereof completelyinhibits the amidolytic activity of FXIIa at a concentration of lessthan 100 nM, more preferably less than 50 nM, even more preferably lessthan 40 nM, or even less than 30 nM. Preferably the antibody orantigen-binding fragment thereof completely inhibits at a concentrationof between 1 pM and 100 nM, more preferably at a concentration between 5pM and 50 nM. Preferably the assay for the amidolytic activity of FXIIais carried out as described in Example 1(5).

Another aspect of the invention is an anti-Factor XII/FXIIa monoclonalantibody or antigen-binding fragment thereof that inhibits FactorXIIa-alpha, preferably human Factor XIIa-alpha, by more than 40%,preferably more than 50%, even more preferably more than 60%, when usedat a molar ratio of FXIIa-alpha to antibody of 1:0.2. Alternatively, theantibody or antigen binding fragment thereof inhibits Factor XIIa-alpha,preferably human Factor XIIa-alpha, by more than 80%, preferably morethan 85%, more preferably more than 90%, at a molar ratio of FXIIa-alphato antibody of 1:0.5; most preferably, the antibody or antigen-bindingfragment thereof achieves complete inhibition of FXIIa-alpha at a molarratio of 1:0.5. Preferably the antibody or antigen-binding fragmentthereof has an affinity to human FXIIa that is at least comparable toantibody 3F7 disclosed herein.

Preferably, the antibody or antigen-binding fragment thereof bindsmurine FXII/FXIIa; more preferably, the level of binding of the antibodyto a polypeptide comprising SEQ ID NO: 2 or relevant fragment thereof inwhich (a) the asparagine residue at position 398 of SEQ ID NO: 2 issubstituted for lysine; or (b) the isoleucine residue at position 438 ofSEQ ID NO: 2 is substituted for alanine, is lower than the level ofbinding of the protein to the corresponding polypeptide comprising SEQID NO: 2 or relevant fragment thereof without said substitution. Arelevant fragment of the polypeptide of SEQ ID NO: 2 comprises thecatalytic center; examples are the light chain, FXIIa-beta, FXIIa-alpha,or the complete FXII.

Preferably, the antibody or antigen-binding fragment thereof comprises aheavy chain variable (vH) region which is more than 85% identical to thesequence of SEQ ID NO: 4, more preferably more than 88%, 90%, 92%, 93%,94%, 95%, 96%, 97%, even more preferably 98%, or even 99% identical tothe sequence of SEQ ID NO: 4. Preferred embodiments of the invention areantibodies or antigen-binding fragments thereof comprising a heavy chainvariable region with the sequence of SEQ ID NOs: 4, 73, 74, 76 or 77.

Preferably, the antibody or antigen binding fragment thereof comprises alight chain variable (vL) region which is more than 85% identical to thesequence of SEQ ID NO: 5, more preferably more than 88%, 90%, 92%, 93%,94%, 95%, 96%, 97%, even more preferably 98%, or even 99% identical tothe sequence of SEQ ID NO: 5. Preferred embodiments of the invention areantibodies or antigen binding fragments thereof comprising a light chainvariable region with the sequence of SEQ ID NOs: 5 or 75.

Preferred embodiments of the invention are antibodies or antigen bindingfragments thereof with a vH region described above combined with a vLregion as described above. Most preferred are antibodies with thefollowing vH/vL combinations:

-   -   (a) A vH region of SEQ ID NO: 4 combined with a vL region of SEQ        ID NO: 5 or SEQ ID NO: 75;    -   (b) A vH region of any of SEQ ID NOs: 4, 73, 74, 76 or 77        combined with a vL region of SEQ ID NO: 5.

Preferably, the antibodies or antigen binding fragments thereof compriseheavy chain CDR1 at least 80% identical to the sequence of SEQ ID NO: 6,preferably heavy chain CDR1 of SEQ ID NO: 6, and/or heavy chain CDR2 atleast 60% identical to the sequence of SEQ ID NO: 7, and/or heavy chainCDR3 at least 80% identical to the sequence of SEQ ID NO: 9. Morepreferably, heavy chain CDR2 has the sequence GIX₁X₂X₃X₄X₅X₆TVYADSVKG(see SEQ ID NO: 8) wherein X₁ is R, N or D, X₂ is P, V, I or M, X₃ is S,P or A, X₄ is G, L, V, or T, X₅ can be any amino acid, preferably X₅ isG, Y, Q, K, R, N or M, and X₆ is T, G, or S, and/or heavy chain CDR3 hasthe sequence ALPRSGYLX₁X₂X₃X₄YYYYALDV (see SEQ ID NO: 10), wherein X₁ isI, M or V, X₂ is S or K, X₃ is P, K, T or H, and X₄ is H, N, G, or Q.Preferably, the antibodies or antigen binding fragments thereof compriselight chain CDR1 at least 50% identical with SEQ ID NO: 11, and/or lightchain CDR2 of SEQ ID NO: 12, and/or light chain CDR3 with the sequenceAX₁WX₂X₃X₄X₅RX₆X₇ (shown in SEQ ID NO: 14), wherein X₁ is A or S, X₅ isL or V, X₆ is G, L, or K, and X₂, X₃, X₄ and X₇ can be any amino acid,preferably X₂ is D, Y, E, T, W, E or S, X₃ is A, N, I, L, V, P, Q, or E,X₄ is S, D, P, E, Q, or R, and X₇ is V, A, D, T, M, or G.

Preferred embodiments of the invention are antibodies or antigen bindingfragments thereof with the heavy chain CDRs described above combinedwith the light chain CDRs as described above.

More preferably, the antibodies or antigen binding fragments thereofcomprise the combinations of heavy chain CDRs (HCDRs) and light chainCDRs (LCDRs) shown in Table 1, wherein the numbers in the columnsunderneath HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are therespective SEQ ID NOs:

mAb HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 6 8 10 11 12 14 3F7 6 7 9 11 1213 VR119 6 29 9 11 12 13 VR112 6 30 9 11 12 13 VR115 6 31 9 11 12 13VR24 6 7 9 44 12 13 VR110 6 32 9 11 12 13 VR107 6 33 9 11 12 13 VR06 6 79 45 12 13 VR31 6 7 9 11 12 52 VR108 6 34 9 11 12 13 VR103 6 35 9 11 1213 VR101 6 36 9 11 12 13 VR16 6 7 9 46 12 13 VR29 6 7 9 11 12 53 VR05 67 9 47 12 13 VR12 6 7 9 48 12 13 VR27 6 7 9 11 12 54 VR10 6 7 9 49 12 13VR149 6 7 39 11 12 13 VR58 6 7 9 11 12 59 VR39 6 7 9 11 12 55 VR167 6 740 11 12 13 VR62 6 7 9 11 12 60 VR109 6 37 9 11 12 13 VR14 6 7 9 50 1213 VR46 6 7 9 11 12 56 VR148 6 7 41 11 12 13 VR159 6 7 42 11 12 13 VR536 7 9 11 12 61 VR52 6 7 9 11 12 62 VR160 6 7 43 11 12 13 VR17 6 7 9 5112 13 VR63 6 7 9 11 12 63 VR41 6 7 9 11 12 57 VR99 6 38 9 11 12 13 VR386 7 9 11 12 58

Preferably, the antibody or antigen binding fragments thereof of theinvention binds human Factor XIIa-beta with a K_(D) of better than10⁻⁷M, more preferably better than 3×10⁻⁸M, more preferably better than10⁻⁸M, even more preferably better than 3×10⁻⁹ M, most preferably 10⁻⁹Mor even 5×10⁻¹⁰M.

Preferably, the antibody or antigen binding fragment thereof of theinvention competes with Infestin, preferably with Infestin-4, forbinding to human Factor XIIa-beta.

The antibody or antigen binding fragment thereof can be any isotype,including IgG, IgM, IgE, IgD, or IgA, and any subtype thereof.Preferably, the antibody or antigen binding fragment thereof of theinvention is a human IgG or variant thereof, preferably human IgG4 orvariant thereof. Methods to switch the type of antibody are well knownin the art. The nucleic acid molecule encoding the v_(H) or v_(L) regionis isolated, and operatively linked to a nucleic acid sequence encodinga different c_(H) or c_(L), respectively, from the constant region of adifferent class of immunoglobulin molecule.

The present disclosure encompasses proteins and/or antibodies describedherein comprising a constant region of an antibody. This includesantigen binding fragments of an antibody fused to a Fc.

Sequences of constant regions useful for producing the proteins of thepresent disclosure may be obtained from a number of different sources.In some examples, the constant region or portion thereof of the proteinis derived from a human antibody. The constant region or portion thereofmay be derived from any antibody class, including IgM, IgG, IgD, IgA andIgE, and any antibody isotype, including IgG1, IgG2, IgG3 and IgG4. Inone example, the constant region is human isotype IgG4 or a stabilizedIgG4 constant region.

In one example, the Fc region of the constant region has a reducedability to induce effector function, e.g., compared to a native orwild-type human IgG1 or IgG3 Fc region. In one example, the effectorfunction is antibody-dependent cell-mediated cytotoxicity (ADCC) and/orantibody-dependent cell-mediated phagocytosis (ADCP) and/orcomplement-dependent cytotoxicity (CDC). Methods for assessing the levelof effector function of an Fc region containing protein are well knownin the art.

In one example, the Fc region is an IgG4 Fc region (i.e., from an IgG4constant region), e.g., a human IgG4 Fc region. Sequences of suitableIgG4 Fc regions will be apparent to the skilled person and/or availablein publically available databases (e.g., available from National Centerfor Biotechnology Information).

In one example, the constant region is a stabilized IgG4 constantregion. The term “stabilized IgG4 constant region” will be understood tomean an IgG4 constant region that has been modified to reduce Fab armexchange or the propensity to undergo Fab arm exchange or formation of ahalf-antibody or a propensity to form a half antibody. “Fab armexchange” refers to a type of protein modification for human IgG4, inwhich an IgG4 heavy chain and attached light chain (half-molecule) isswapped for a heavy-light chain pair from another IgG4 molecule. Thus,IgG4 molecules may acquire two distinct Fab arms recognizing twodistinct antigens (resulting in bispecific molecules). Fab arm exchangeoccurs naturally in vivo and can be induced in vitro by purified bloodcells or reducing agents such as reduced glutathione. A “half antibody”forms when an IgG4 antibody dissociates to form two molecules eachcontaining a single heavy chain and a single light chain.

In one example, a stabilized IgG4 constant region comprises a proline atposition 241 of the hinge region according to the system of Kabat (Kabatet al., Sequences of Proteins of Immunological Interest Washington D.C.United States Department of Health and Human Services, 1987 and/or1991). This position corresponds to position 228 of the hinge regionaccording to the EU numbering system (Kabat et al., Sequences ofProteins of Immunological Interest Washington D.C. United StatesDepartment of Health and Human Services, 2001 and Edelman et al., Proc.Natl. Acad. Sci. USA, 63, 78-85, 1969). In human IgG4, this residue isgenerally a serine. Following substitution of the serine for proline,the IgG4 hinge region comprises a sequence CPPC. In this regard, theskilled person will be aware that the “hinge region” is a proline-richportion of an antibody heavy chain constant region that links the Fc andFab regions that confers mobility on the two Fab arms of an antibody.The hinge region includes cysteine residues which are involved ininter-heavy chain disulfide bonds. It is generally defined as stretchingfrom Glu226 to Pro243 of human IgG1 according to the numbering system ofKabat. Hinge regions of other IgG isotypes may be aligned with the IgG1sequence by placing the first and last cysteine residues forminginter-heavy chain disulphide (S—S) bonds in the same positions (see forexample WO2010/080538).

Additional examples of stabilized IgG4 antibodies are antibodies inwhich arginine at position 409 in a heavy chain constant region of humanIgG4 (according to the EU numbering system) is substituted with lysine,threonine, methionine, or leucine (e.g., as described in WO2006/033386).The Fc region of the constant region may additionally or alternativelycomprise a residue selected from the group consisting of: alanine,valine, glycine, isoleucine and leucine at the position corresponding to405 (according to the EU numbering system). Optionally, the hinge regioncomprises a proline at position 241 (i.e., a CPPC sequence) (asdescribed above).

In another example, the Fc region is a region modified to have reducedeffector function, i.e., a “non-immunostimulatory Fc region”. Forexample, the Fc region is an IgG1 Fc region comprising a substitution atone or more positions selected from the group consisting of 268, 309,330 and 331. In another example, the Fc region is an IgG1 Fc regioncomprising one or more of the following changes E233P, L234V, L235A anddeletion of G236 and/or one or more of the following changes A327G,A330S and P331S (Armour et al., Eur J. Immunol. 29:2613-2624, 1999;Shields et al., J Biol. Chem. 276(9):6591-604, 2001). Additionalexamples of non-immunostimulatory Fc regions are described, for example,in Dall'Acqua et al., J. Immunol. 177: 1129-1138, 2006; and/or Hezareh JVirol; 75: 12161-12168, 2001).

In another example, the Fc region is a chimeric Fc region, e.g.,comprising at least one C_(H)2 domain from an IgG4 antibody and at leastone C_(H)3 domain from an IgG1 antibody, wherein the Fc region comprisesa substitution at one or more amino acid positions selected from thegroup consisting of 240, 262, 264, 266, 297, 299, 307, 309, 323, 399,409 and 427 (EU numbering) (e.g., as described in WO2010/085682).Exemplary substitutions include 240F, 262L, 264T, 266F, 297Q, 299A,299K, 307P, 309K, 309M, 309P, 323F, 399S, and 427F.

The present disclosure also contemplates additional modifications to anantibody.

For example, the antibody comprises one or more amino acid substitutionsthat increase the half-life of the protein. For example, the antibodycomprises a Fc region comprising one or more amino acid substitutionsthat increase the affinity of the Fc region for the neonatal Fc region(FcRn). For example, the Fc region has increased affinity for FcRn atlower pH, e.g., about pH 6.0, to facilitate Fc/FcRn binding in anendosome. In one example, the Fc region has increased affinity for FcRnat about pH 6 compared to its affinity at about pH 7.4, whichfacilitates the re-release of Fc (and therefore of Fc region-comprisingmolecules) into blood following cellular recycling. These amino acidsubstitutions are useful for extending the half life of a protein, byreducing clearance from the blood.

Exemplary amino acid substitutions include T250Q and/or M428L or T252A,T254S and T266F or M252Y, S254T and T256E or H433K and N434F accordingto the EU numbering system. Additional or alternative amino acidsubstitutions are described, for example, in US20070135620 or US7083784.

More preferably, the antibody of the invention is a human IgG1 or humanIgG4, engineered for enhanced binding to the human neonatal Fc receptorFcRn at a lower pH, e.g. pH 6, which leads to an increased half life ofthe antibody in human serum. Methods to screen for optimal Fc variantsfor optimizing FcRn binding have been described (e.g. Zalevsky et al(2010) Nature Biotech 28, 157-159).

Other preferred antibodies or antigen binding fragments thereof of theinvention comprise mammalian immunoglobulin constant regions, such asthe constant regions of mammalian isotypes such as IgG, IgM, IgE, IgD,or IgA, and any subtype thereof. Preferably, the antibody is a mammalianIgG, including mouse IgG, pig IgG, cow IgG, horse IgG, cat IgG, dog IgGand primate IgG or variants thereof. These antibodies may be chimericantibodies, where the human variable regions of the invention arecombined with the constant region of the immunoglobulin of the selectedspecies. Alternatively, the antibody or antigen binding fragmentsthereof may be produced by grafting the human CDR regions describedherein into the framework residues from an immunoglobulin of theselected species.

Preferably the antibodies or antigen binding fragments thereof of theinvention are in their mature form, i.e. without the signal peptide;however, the antibodies or antigen binding fragments thereof includingthe signal peptides are also included in the invention.

The antigen binding fragment may be any fragment of an antibody of theinvention that maintains the ability to bind FXIIa. Preferred antigenbinding fragments are an Fab fragment, an Fab′ fragment, an F(ab′)₂fragment, an Fv fragment, a single chain antibody, a single chain Fvfragment, a disulfide stabilized Fv protein, or a dimer of a singlechain Fv fragment. Antibodies also included in the invention are achimeric antibody, a humanized antibody, a murinized antibody or abispecific antibody. Methods for producing these fragments andantibodies are well known in the art (see for example, Harlow & Lane:Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).

Also included in the invention is a fusion protein or a phage particlecomprising the antigen binding fragment of the antibody of theinvention. The antigen binding fragment may, for example, be fused withhuman serum albumin or a variant thereof. The skilled person will bewell aware of other proteins that can be used as fusion partners forantigen binding fragments. The antibody or antigen binding fragmentthereof may also be fused to a tag, such as a hexa-Histidine tag. Thetag may be provided with a cleavable linker peptide, so that it can beremoved from the antibody or antigen binding fragment thereof whendesired.

Another aspect of the invention is a nucleic acid encoding the antibody,or antigen-binding fragment thereof, of the invention. Preferably, thenucleic acid also comprises a region encoding a signal peptide,preferably the nucleic acid comprises a region encoding a signal peptidefor the heavy chain and a region encoding a signal peptide for the lightchain.

Nucleic acid molecules encoding the polypeptides provided by theinvention can be readily produced by the skilled person, using the aminoacid sequences provided, the genetic code and sequences available inpublic databases. In addition, a variety of functionally equivalentnucleic acids can be readily produced and are therefore also included inthe present invention. The nucleic acid molecules can be prepared by anysuitable method, for example by direct chemical synthesis. Methods forpreparing DNA are well known in the art.

Yet another aspect of the invention is a vector comprising the nucleicacid encoding the antibody, or antigen-binding fragment thereof, of theinvention, operably linked to a suitable promoter sequence orincorporated into a suitable expression cassette, which may includeadditional regulatory elements such as enhancer elements to increaseexpression levels. Preferably a strong promoter is used. For expressionin E. coli, a promoter such as T7, lac, trp or lambda promoters may beused, preferably in conjunction with a ribosome binding site and atranscription termination signal. For mammalian cells, SV40, CMV orimmunoglobulin promoters can be used to provide high expression levels.Preferably, the vector is a mammalian cell expression vector, morepreferably a vector selected from Lonza's GS System™ or Selexis GeneticElements™ systems. Preferably, the vector also contains a selectablemarker sequence such as gpt, neo, amp or hyg genes, and a geneamplification system such as glutamine synthetase or DHFR. Anotherpreferred vector is a yeast expression vector, e.g. an expression vectoroptimized for Pichia pastoris. The vector may also be a viral vector,e.g. a vector based on vaccinia virus, adenovirus, or a retrovirus. Thevector may also be a baculovirus for expression in insect cells.

A further aspect of the invention is a cell line or yeast cellcomprising the vector of the invention. Preferably the cell line is amammalian cell line, such as CHO, HEK293, MDCK, COS, HeLa, or myelomacell lines such as NSO. Another embodiment is an insect cell line foruse with a baculovirus, such as SF9 cells, SF21 cells, or HighFive™cells. Yet another cell is a yeast cell, such as Saccharomyces, e.g. S.cerevisiae, or Pichia pistoris. Bacterial host cells such as E. coli arealso possible. Methods for introducing DNA into the respective hostcells are well known in the art. For example, when the host cell is amammalian cell line, techniques such as lipofection or electroporationmay be used.

Another aspect of the invention is a method of producing the antibody orantigen binding fragment thereof of the invention, comprising culturingthe host cells, such as the cell line or yeast cell, of the inventionunder appropriate conditions to express the antibody or antigen bindingfragment thereof. The antibody of antigen binding fragment thereof maythen be purified. Preferably, the antibody or antigen binding fragmentthereof is secreted by the host cell, and can then easily be purifiedfrom the culture supernatant. Techniques for purifying antibodies arewell known in the art, and include techniques such as ammonium sulfateprecipitation, size exclusion chromatography, affinity chromatography,ion exchange chromatography and others.

When expressed in E. coli, the antibodies or antigen binding fragmentsthereof may be produced in inclusion bodies. Methods to isolateinclusion bodies and refold the expressed protein are well known in theart.

Yet another aspect of the invention is the antibody or antigen-bindingfragment thereof of the invention for medical use.

A further aspect of the invention is the antibody or antigen-bindingfragment thereof for use in the prevention of the formation and/or thestabilization of thrombi in a human or animal subject. Three-dimensionalintraluminal thrombus growth is reduced or even prevented. Thus, thisaspect of the invention relates to the antibody or antigen-bindingfragment thereof for use in the treatment or prevention of a disorderselected from the group consisting of venous, arterial or capillarythrombus formation, thrombus formation in the heart, thrombus formationduring and/or after contacting blood of a human or animal subject withartificial surfaces and thromboembolism by preventing and/or treatingthe formation and/or stabilization of thrombi and thereby thethree-dimensional intraluminal thrombus growth. Yet another aspect ofthe invention is the antibody or antigen binding fragment thereof foruse in the treatment of intraluminal thrombi in a human or animalsubject related to a disorder selected from the group consisting ofvenous, arterial or capillary thrombus formation, thrombus formation inthe heart, thrombus formation during and/or after contacting blood of ahuman or animal subject with artificial surfaces or thromboembolism.Preferably, the venous or arterial thrombus formation is stroke,myocardial infarction, deep vein thrombosis, portal vein thrombosis,thromboembolism, renal vein thrombosis, jugular vein thrombosis,cerebral venous sinus thrombosis, Budd-Chiari syndrome orPaget-Schroetter disease.

A further aspect of the invention relates to the antibody or antigenbinding fragment thereof for use in the prevention and/or treatment ofinflammation, a neurological inflammatory disease, interstitial lungdisease, complement activation, fibrinolysis, angiogenesis and diseasesrelated to FXII/FXIIa-induced kinin formation or FXII/FXIIa-mediatedcomplement activation. Preferably, the diseases related toFXIIFXIIa-induced kinin formation are selected from the group hereditaryangioedema, bacterial infections of the lung, trypanosoma infections,hypotensive shock, pancreatitis, chagas disease, articular gout,arthritis, disseminated intravascular coagulation (DIC) and sepsis.

Preferably the interstitial lung disease is fibroproliferative and/oridiopathic pulmonary fibrosis.

An aspect of the invention is also a method of treatment of any of theconditions or diseases mentioned above in a subject, by administering tothe subject in need thereof a therapeutically effective amount of theantibody or antigen-binding fragment thereof.

The beneficial effect of the antibody or antigen-binding fragmentthereof in the various conditions can be verified, for example, byemploying a suitable animal model, for example a mouse model. Bycomparison of animals treated with the antibody or antigen-bindingfragment thereof and a control group, the beneficial effect of thetreatment of the respective disease with the antibody can bedemonstrated. Alternatively, patient plasma samples can be tested forrelevant parameters. For example, a beneficial effect in treating orpreventing disease-related symptoms in patients with hereditaryangioedema can be tested by employing a mouse model, for example asdescribed in Han et al (2002) J. Clin. Invest. 109:1057-1063. An invitro test, using patient plasma samples can also be envisaged; treatedand untreated patient plasma samples could be compared for bradykininand/or high molecular weight kininogen levels. The antibody shouldreduce the bradykinin generation, and/or prevent a decrease in highmolecular weight kininogen levels.

Preferably, the thrombus formation occurs during and/or after contactingblood of a human or animal subject with artificial surfaces duringand/or after a medical procedure performed on said human or animalsubject and said antibody or antigen binding fragment thereof isadministered before and/or during and/or after said medical procedure,and further wherein

-   -   (i) the artificial surface is exposed to at least 80% of the        blood volume of the subject and the artificial surface is at        least 0.2 m² or    -   (ii) the artificial surface is a container for collection of        blood outside the body of the subject or    -   (iii) the artificial surface is a stent, valve, intraluminal        catheter, or a system for internal assisted pumping of blood.

Preferably, the bleeding risk of said human or animal subject

-   -   (i) is not increased; and/or    -   (ii) is determined        -   a) via the ear or finger tip bleeding time according to Duke            and wherein said ear or finger tip bleeding time is not            longer than 10 minutes or        -   b) according to the method of Ivy and wherein the bleeding            time is not longer than 10 minutes or        -   c) according to the method of Marx and the bleeding time is            not longer than 4 minutes.

The medical procedure may be

-   -   i) any procedure requiring a cardiopulmonary bypass or    -   ii) the oxygenation of blood via extracorporeal membrane        oxygenation or    -   iii) the internal assisted pumping of blood or    -   iv) the dialysis of blood or    -   v) the extracorporeal filtration of blood or    -   vi) the collection of blood in any repository for later use in        an animal or a human subject or    -   vii) the use of intraluminal catheter(s) or    -   viii) the use of stent(s) or    -   ix) the use of artificial heart valve(s).

The antibody or antigen-binding fragment thereof of the invention may beadministered before, after and/or during a medical procedure requiringcardiopulmonary bypass, or a medical procedure comprising the collectionof blood in any repository for later use in an animal or human subject.It may also be administered by being coated on the artificial surface.Where the medical procedure involves blood donation, the antibody orantigen-binding fragment thereof may be:

-   -   i) administered to the blood donor before and/or during the        blood donation process or    -   ii) mixed with the blood in the collection repository or    -   iii) administered to the blood recipient before, during, and/or        after the blood is administered to the human or animal        recipient.

Preferably the amount of heparin or derivatives thereof and/or hirudinor derivatives thereof which is added in addition to the antibody orantigen-binding fragment thereof before and/or during and/or after themedical procedure is reduced or even completely omitted as compared tothe amount of heparin or derivatives thereof and/or hirudin orderivatives thereof which is administered normally before and/or duringsaid medical procedure when no said anti-FXII/FXIIa antibody or antigenbinding fragment thereof is administered.

Preferably, the prothrombotic risk following the postoperativeantagonism of heparin or derivatives thereof and/or the postoperativeantagonism of hirudin or derivatives thereof is prevented or reduced;the prothrombotic risk may also be caused by the administration ofprotamine.

A further aspect of the invention is the antibody or antigen-bindingfragment thereof of the invention for the prevention or the treatment ofPump Head syndrome.

Yet a further aspect of the invention is a medical device coated with anantibody or antigen-binding fragment thereof of the invention, whereinthe device is a cardiopulmonary bypass machine, an extracorporealmembrane oxygenation system for oxygenation of blood, a device forassisted pumping of blood, a blood dialysis device, a device for theextracorporeal filtration of blood, a repository for use in thecollection of blood, an intraluminal catheter, a stent, an artificialheart valve, and/or accessories for any one of said devices includingtubing, cannulae, centrifugal pump, valve, port, and/or diverter.

Another aspect of the invention is the antibody or antigen-bindingfragment thereof for use for administration in a patient receiving amedical procedure, wherein the medical procedure comprises contact withat least one of:

-   -   (a) heart,    -   (b) at least one blood vessel chosen from: the aorta, the aortic        arch, a carotid artery, a coronary artery, brachiocephalic        artery, vertebrobasilar circulation, intracranial arteries,        renal artery, a hepatic artery, a mesenteric artery, and/or a        blood vessel of the arterial system cranial to the heart,    -   (c) a venous blood vessel if the patient has a known septal        defect;        and wherein the medical procedure comprises release of at least        one embolus in at least one of said blood vessels in the body        that could result in ischemia in at least one target organ and        administration of the antibody or antigen-binding fragment        thereof before, during, and/or after the medical procedure.

The embolus may be comprised of bubbles, oil, fat, cholesterol,coagulated blood, and/or debris.

The target organ may be:

-   -   (a) brain, and wherein the patient has, has had, or is at risk        for:        -   (i) silent brain ischemia or        -   (ii) a stroke caused by a nonthrombolysable substance;            and/or    -   (b) heart, kidney, liver; and/or gastrointestinal tract organ.

Preferably, the medical procedure comprises contact with the inside ofor clamping of at least one or more of said blood vessels.

Preferably, the medical procedure is a vascular procedure that comprisesany one or more of a catheter, a stent, a balloon, a graft, and/oradministering a contrast agent.

Preferably, the medical procedure is a vascular surgery and/or is avascular procedure that is diagnostic. More preferably, the medicalprocedure is coronary angiography, carotid artery stenting, percutaneouscoronary intervention, carotid endarerectomy, a cardiovascular surgery,or dilation of stenotic renal artery.

Another aspect of the invention is the antibody or antigen-bindingfragment thereof for use in the prevention or treatment of a conditionassociated with increased vascular permeability, in particular increasedretinal vascular permeability, including progressive retinopathy,sight-threatening complication of retinopathy, macular edema,non-proliferative retinopathy, proliferative retinopathy, retinal edema,diabetic retinopathy, hypertensive retinopathy, and retinal trauma.

Another aspect of the invention is a pharmaceutical compositioncomprising the antibody or antigen-binding fragment thereof of theinvention. The antibody or antigen-binding fragment thereof can beformulated according to known methods for preparing a pharmaceuticalcomposition. For example, it can be mixed with one or morepharmaceutically acceptable carriers, diluents or excipients. Forexample, sterile water or physiological saline may be used. Othersubstances, such as pH buffering solutions, viscosity reducing agents,or stabilizers may also be included.

A wide variety of pharmaceutically acceptable excipients and carriersare known in the art. Such pharmaceutical carriers and excipients aswell as suitable pharmaceutical formulations have been amply describedin a variety of publications (see for example “PharmaceuticalFormulation Development of Peptides and Proteins”, Frokjaer et al.,Taylor & Francis (2000) or “Handbook of Pharmaceutical Excipients”,3^(rd) edition, Kibbe et al., Pharmaceutical Press (2000) A. Gennaro(2000) “Remington: The Science and Practice of Pharmacy”, 20^(th)edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Formsand Drug Delivery Systems (1999) H. C. Ansel et al., eds 7^(th) ed.,Lippincott, Williams, & Wilkins; and Handbook of PharmaceuticalExcipients (2000) A. H. Kibbe et al., eds., 3^(rd) ed. Amer.Pharmaceutical Assoc). In particular, the pharmaceutical compositioncomprising the antibody of the invention may be formulated inlyophilized or stable soluble form. The polypeptide may be lyophilizedby a variety of procedures known in the art. Lyophilized formulationsare reconstituted prior to use by the addition of one or morepharmaceutically acceptable diluents such as sterile water for injectionor sterile physiological saline solution.

The pharmaceutical composition of the invention can be administered indosages and by techniques well known in the art. The amount and timingof the administration will be determined by the treating physician orveterinarian to achieve the desired purposes. The route ofadministration can be via any route that delivers a safe andtherapeutically effective dose to the blood of the subject to betreated. Possible routes of administration include systemic, topical,enteral and parenteral routes, such as intravenous, intraarterial,subcutaneous, intradermal, intraperitoneal, oral, transmucosal,epidural, or intrathecal. Preferred routes are intravenous orsubcutaneous.

The effective dosage and route of administration are determined byfactors such as age and weight of the subject, and by the nature andtherapeutic range of the antibody or antigen-binding fragment thereof.The determination of the dosage is determined by known methods, no undueexperimentation is required.

A therapeutically effective dose is a dose of the antibody or antigenbinding fragment thereof of the invention that brings about a positivetherapeutic effect in the patient or subject requiring the treatment. Atherapeutically effective dose is in the range of about 0.01 to 50mg/kg, from about 0.01 to 30 mg/kg, from about 0.1 to 30 mg/kg, fromabout 0.1 to 10 mg/kg, from about 0.1 to 5 mg/kg, from about 1 to 5mg/kg, from about 0.1 to 2 mg/kg or from about 0.1 to 1 mg/kg. Thetreatment may comprise giving a single dose or multiple doses. Ifmultiple doses are required, they may be administered daily, every otherday, weekly, biweekly, monthly, or bimonthly or as required. Adepository may also be used that slowly and continuously releases theantibody or antigen-binding fragment thereof. A therapeuticallyeffective dose may be a dose that inhibits FXIIa in the subject by atleast 50%, preferably by at least 60%, 70%, 80%, 90%, more preferably byat least 95%, 99% or even 100%.

A further aspect of the invention is an affinity-matured antibody orantigen-binding fragment thereof of the antibodies (or antigen bindingfragments thereof) described above.

DEFINITIONS

Unless otherwise stated, all terms are used according to conventionalusage.

“Antibody” in its broadest sense is a polypeptide comprising animmunoglobulin variable region which specifically recognizes an epitopeon an antigen. Antibodies are usually comprised of two identical heavychains and two identical light chains, each of which has a variableregion at its N-terminus (v_(H) and v_(L) region). Usually a vH and a vLregion will combine to form the antigen binding site. However, singledomain antibodies, where only one variable region is present and bindsto the antigen, have also been described.

Typically, an antibody contains two heavy and two light chains,connected by disulfide bonds. There are 5 major isotypes of antibodies(IgG, IgM, IgE, IgA, IgD), some of which occur as multimers of the basicantibody structure. The isotype is determined by the constant region ofthe heavy chains. There are two types of light chains, lambda and kappa.

The term “antibody” as used herein includes intact antibodies, as wellas variants and portions thereof that retain antigen binding. Thisincludes fragments of antibodies such as Fab fragments, F(ab′)₂fragments, Fab′ fragments, single chain Fv fragments, ordisulfide-stabilized Fv fragments. Thus, the term “antibody orantigen-binding fragment thereof” in this document is onlyprecautionary, the term “antibody” alone is already intended to coverthe antibody and antigen-binding fragments thereof.

Each heavy and light chain consists of a variable region and a constantregion. The variable regions contain framework residues andhypervariable regions, which are also called complementarity determiningregions or CDRs. The extent of the framework residues and CDRs isdetermined according to Kabat; the Kabat database is available online(Kabat E A, Wu T T, Perry H M, Gottesman K S, FoeIler C (1991) Sequencesof proteins of immunological interest, 5^(th) edn. U.S. Department ofHealth and Human services, NIH, Bethesda, Md.). The CDR regions areimportant in binding to the epitope and therefore determine thespecificity of the antibody.

A “monoclonal antibody” is an antibody produced by a single clone of Blymphocytes, or by a cell line engineered to express a single antibody.

A “chimeric antibody” is an antibody with the variable regions from onespecies grafted onto the constant regions from a different species. A“humanized” antibody is an antibody where CDR regions from a differentspecies, e.g. a mouse monoclonal antibody, are grafted into theframework of a human antibody. Analogously, a “murinized” antibody is anantibody where the CDR regions from a different species, e.g. a humanmonoclonal antibody, are grafted into the framework of a mouse antibody.A human antibody is an antibody that is wholly derived from human, i.e.human CDRs in a human framework and any constant region suitable foradministration to a human.

A “germlined” antibody is an antibody where somatic mutations thatintroduced changes into the framework residues are reversed to theoriginal sequence present in the genome.

“Antigen binding fragment” refers to any fragment of an antibody thatretains the ability to specifically bind the epitope of the antigen thatthe antibody binds to. These include but are not limited to Fab,F(ab′)₂, or single chain Fv fragments.

“Binding affinity” refers to the affinity of the antibody to itsantigen. It can be measured by a variety of techniques, e.g. surfaceplasmon resonance based technology (BiaCore).

“Epitope” is the antigenic determinant, it is defined by the residues orparticular chemical structures that the antibody makes contact with onthe antigen.

“Sequence identity” relates to the similarity of amino acid sequences.The best possible alignment of two sequences is prepared, and thesequence identity is determined by the percentage of identical residues.Standard methods are available for the alignment of sequences, e.g.algorithms of Needleman and Wunsch (J Mol Biol (1970) 48, 443), Smithand Waterman (Adv Appl Math (1981) 2, 482), Pearson and Lipman (ProcNatl Acad Sci USA (1988) 85, 2444), and others. Suitable software iscommercially available, e.g. the GCG suite of software (Devereux et al(1984), Nucl Acids Res 12, 387), where alignments can be produced using,for example, GAP or BESTFIT with default parameters, or successorsthereof. The Blast algorithm, originally described by Altschul et al (J.Mol. Biol. (1990) 215, 403), but further refined to include gappedalignments (Blast 2), available from various sources such as the EBI,NCBI, will also produce alignments and calculate the % identity betweentwo sequences.

“Specific binding” refers to the binding to substantially only a singleantigen.

“FXII/FXIIa” refers to either or both of Factor XII and activated FactorXII (FXIIa). Thus “FXII/FXIIa inhibitor” includes inhibitors of eitheror both of FXII and FXIIa. Further, anti-FXII/FXIIa antibodies includeantibodies that bind to and inhibit either or both of FXII and FXIIa.

“Infestins” are a class of serine protease inhibitors derived from themidgut of the hematophagous insect, Triatoma infestans, a major vectorfor the parasite Trypanosoma cruzi, known to cause Chagas' disease(Campos I T N et al. 32 Insect Biochem. Mol. Bio. 991-997, 2002; CamposI T N et al. 577 FEBS Lett. 512-516, 2004). This insect uses theseinhibitors to prevent coagulation of ingested blood. The Infestin geneencodes 4 domains that result in proteins that can inhibit differentfactors in the coagulation pathway. In particular, domain 4 encodes aprotein (Infestin-4) that is a strong inhibitor of FXIIa. Infestin-4 hasbeen administered in mice without bleeding complications (WO2008/098720). Infestin-4 has been coupled to human serum albumin(rHA-Infestin-4).

“Complete inhibition of the amidolytic activity of FXIIa” means aninhibition of 80% or more, preferably of 90% or more, more preferably of95% or more, of the activity observed in a control experiment withoutany inhibitor present. “Activity of Factor XIIa” includes the activityof all forms of Factor XIIa, such as FXIIa-alpha and FXIIa-beta.

The terms “treatment” or “treating” or “therapy” are intended to beinterpreted broadly; an improvement in any disease-related symptom inthe subject or patient or in a level of a relevant biomarker would beincluded.

EXAMPLES

The following examples illustrate certain embodiments of the inventionbut are not intended to limit the invention to the embodiments that areexemplified. The techniques used are based on standard laboratoryprocedures well known to the skilled person, and described in standardlaboratory manuals.

Aim

To isolate fully human antibodies from the DYAX Fab-based phage displaylibrary which are able to effectively inhibit the amidolytic activity ofhuman FXIIa.

Materials

rHA-Infestin-4 (inhibitor of FXIIa amidolytic activity) was supplied byDrs. Thomas Weimer, Holger Lind, and Stefan Schmidbauer (CSL Behring).Human FXII, FXIIa, and FXIIa beta were purchased from Enzyme ResearchLaboratories (supplied by Banksia Scientific, Qld, Australia).Chromogenic substrate S-2303 was from Chromogenix (supplied by AbacusALS). Sulfo-NHS-SS-Biotin and TMB Substrate Solution were from Pierce.Enzymes and M13-KO7 helper phage were from New England Biolabs. Maxisorpimmunoplates were from Nunc. Dynabeads M-280 Streptavidin were fromInvitrogen Corp. Twin tec skirted 96-well PCR plates were fromEppendorf. Taq DNA polymerase was from Scientifix. ExoSAP-It wassupplied by GE Healthcare. BigDye Terminator sequencing kit was fromApplied Biosystems. Anti-human FXII antibody (OT-2) was from Sanquin(Amsterdam, Netherlands).

Example 1 Phage Display Selection 1) Phage Panning Method

A human Fab-based phage display library (Dyax Corp. Cambridge, Mass.)was used to screen against biotinylated FXIIa beta. Prior to initiatingeach round of selection, the antibody library was preincubated with 500μL of 4% milk in PBS for 1 hr at room temperature (RT). 100 μL aliquotsof M280 Streptavidin beads were coated with 3 μg of biotinylated FXIIabeta overnight at 4° C., followed by washing 3 times in PBS/0.05% Tween20 (PBST) and once in PBS using a KingFisher magnetic particle processor(Thermo Fisher Scientific). Beads were collected using a Dynal magneticparticle separator (MPS) (Invitrogen Corp.), resuspended in 1 mL of 2%milk in PBS, and tumbled at RT for 1 hr. Blocked beads were collectedusing the MPS and Round 1 was performed by incubating 5.5×10¹² colonyforming units (cfu) of phage with immobilised FXIIa beta in total volumeof 1 mL at RT for 20 minutes. Following the incubation the beads werecollected and washed 10 times with PBST using the Kingfisher, followedby 2 manual washes in PBS. Finally, the beads were resuspended in 500 μLPBS and designated as Round 1 output (approximately 0.5×10⁸ cfu total).The Round 1 output phage were then amplified by infecting 6 mls of TG1culture with one half (250 μL) of beads at 37° C. for 30 minutes, withshaking at 250 rpm. One mL of infected culture was removed and stored at4° C., and 2.5×10¹⁰ pfu of M13KO₇ helper phage were added to theremaining 5 mLs of culture, followed by an additional incubation at 37°C. without shaking. The amplification was completed by addition of 30mLs of 2xYT media (containing 100 μg/mL Ampicillin and 50 μg/mLKanamycin) and an overnight incubation at 30° C. Followingamplification, the bacterial pellets were harvested by centrifugationfor 30 min at 4000 rpm, and the phage were precipitated from theresulting medium following the addition of 1:5 volume NaCl-PEG solution(20% PEG 8000, 2.5 M NaCl) and incubation on ice for 60 min. Theprecipitate was resuspended in 1 mL PBS, bacterial debris removed bycentrifugation at 8000 rpm using a bench top centrifuge for 10 minutesand the phage precipitated again as described above. The final phagepellets were resuspended in a total volume of 1 mL in PBS, and titeredto be used as input for the next round of selection. Rounds 2 and 3 wereperformed as described for Round 1. Following Round 3, a pilot scaleselection of clones and preliminary analysis for binding to FXIIa betawas done by ELISA.

2) Pilot Scale Picking and ELISA Analysis of Clones from Round 3 ofPhage Display Selection

The preliminary screening of Round 3 output clones was carried out byFab-phage ELISA. Colonies were picked and inoculated into 120 μL of 2xYTmedium, containing 2% glucose and 100 μg/mL ampicillin. These wereshaken overnight at 37° C., 250 rpm (Infors Supershaker) and designated“masterplate”. These cultures were used to inoculate 100 μL of 2xYT/100μg/mL ampicillin in deep well plates, and plates incubated at 37° C.,700 rpm to an OD600 of approximately 0.5. 100 μL of helper phage wasthen added to a final concentration of 0.5×10¹⁰ pfu, and platesincubated without shaking for 30 min at 37° C. 2xYT media (containing100 μg/mL Ampicillin and 100 μg/mL Kanamycin) was added to the rescuedcultures to give a final concentration of 25 μg/mL of kanamycin,followed by an overnight incubation at 30° C. with shaking (650 rpm).The resultant cultures were spun at 600 g for 30 minutes, andsupernatants used for phage ELISA.

For Fab-phage ELISA, Nunc immunoplates were coated overnight at 4° C.with 100 μL/well of 1 μg/mL FXIIa in PBS. Negative control wells coatedwith PBS alone were also included. Wells were then blocked for 2 hrs at37° C. with 200 μL of 5% skim milk/PBS, and washed 3× in PBST. Fifty μLof 1% skim milk/PBST and 50 μL of phage culture supernatant were addedto each well, and plates were incubated with shaking at room temperaturefor 2 hrs. Plates were than manually washed 5 times with PBST, and 100μL of anti-M13 mAb diluted 1/5000 in 1% milk/PBST was added to eachwell, followed by 30 min incubation at RT with shaking. Plates were thenwashed as before, and 100 μL of TMB substrate was added to each well andthe plates then incubated for 10 minutes at RT with shaking. Thereaction was stopped by the addition of 50 μL of 2M phosphoric acid, andthe absorbance was read at 450 nm in a microplate reader (WallacVictor). Twelve clones appeared positive in the single well ELISA, andwere further tested in a competition ELISA.

3) Analysis of Clones from Round 3 of Selection: Competition Phage ELISA

The twelve clones found reactive to FXIIa in a single well Fab-phageELISA were further tested for reactivity to FXIIa in a competitionELISA. Briefly, the phage titres from culture supernatants (see previoussection) were first determined using a titration ELISA. For titrationELISA, Nunc immunoplates were coated overnight at 4° C. with 100 μL/wellof 1 μg/mL FXIIa in PBS. Negative control wells coated with PBS alonewere also included. Wells were then blocked for 2 hrs at 37° C. with 200μL of 5% skim milk/PBS, and washed 3× in PBS/0.05% Tween 20 (PBST).Fifty μL of phage supernatants were 4-fold serially diluted in 1% skimmilk/PBST, and 100 μL of each dilution were added to the blocked plate.After 1.5 hr incubation at RT with shaking, plates were manually washed5 times in PBST, and the rest of the ELISA protocol was followedessentially as described in previous section. The data was plotted usingKaleidaGraph software with Sigmoidal curve fit, and EC₅₀ value wasrecorded.

For competition ELISA, Nunc 96-well immunoplates were coated and blockedas above. Phage concentrations were fixed at a level determined from thetitration ELISA, and the competitor protein (rHA-Infestin-4) wasserially diluted. Briefly, 4-fold serial dilutions of the competitorprotein were made by having 100 μL of 2 times competitor in the initialwell (ie. 200 nM for desired 100 nM concentration) with 75 μL dilutionbuffer (1% skim milk/PBST) in remaining wells, and serially diluting 25μL of competitor down the plate. 75 μL of 2× phage stock (dilutiondetermined from titration ELISA) were added to each well, and 100 μLfrom each well were transferred into a coated and blocked plate, and therest of ELISA protocol was followed as described above. Phage expressingInfestin domain 4 (Inf4) as a gene-III fusion were used as positivecontrol in the competition ELISA. Phage clones designated 3F7 and 3H4showed competition with EC₅₀ values equivalent to the controlInf4-phage, and were selected for further analysis (FIG. 1). The resultsof the competition ELISA indicate that rHA-Infestin-4 is able to competewith 3F7 and 3H₄Fab-phage and most likely bind to similar regions onFXIIa. All other phage clones whilst able to bind to FXIIa were notcompeted by rHA-infestin-4 (as represented by clone 3G5 in FIG. 1) andhence were unlikely to bind to similar regions within the catalyticdomain of FXIIa.

4) Analysis of Clone 3F7: Sequence Analysis

To determine the amino acid sequences for Fab clones 3F7 and 3H4, 5 mLovernight cultures were started using 5 μL of “masterplate” cultures,and plasmids were isolated using Qiagen miniprep kit. The Fab casetteDNA was sequenced using CH₁Rev and pLacPCRfw primers (Table 2).Sequencing reactions and electrophoresis were carried out at the DNAsequencing facility of Department of Pathology, Melbourne University.The sequences were analyzed using SeqMan (Lasergene), and found to be100% identical, hence a single antibody (3F7) with the ability tocompete with infestin-4 for binding to FXIIa was obtained from panning.

TABLE 2  Sequencing primers used for thecharacterization of phage clones Primer SEQ name Sequence ID NO CH1 Rev5′ GTCCTTGACCAGGCAGCCCAG 3′ 64 pLacPCRfw 5′ GTGAGTTAGCTCACTCATTAG 3′ 65wt GIII 5′ TTTTCATCGGCATTTTCGGTC 3′ 66 stump rev KpaCLfwd 5′CCATCTGATGAGCAGTTGAAATCT 3′ 67 LdaCLfwd 5′ GTTCCCGCCCTCCTCTGAGGAGCT 3′68 PUCrev 5′ AGCGGATAACAATTTCACACAGG 3′ 69 3254 5′GGTTCTGGCAAATATTCTG 3′ 70 Seq CL 5′ GTTGCACCGACCGAATGTA 3′ 71 lambdaSeq CH1 5′ ACCGTGAGCTGGAACAGCGGTGCGC 3′ 72

TABLE 3  Sequences of the variable regions and CDRs of3F7. CDR′s defined according to KABAT numberingsystem (Kabat EA, Wu TT, Perry HM, Gottesman KS, Foeller C (1991) Sequences of proteins of immunological interest, 5^(th) edn. U.S. Departmentof Health and Human services, NIH, Bethesda, MD) RegionAmino acid sequenc vH EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYIMQWVRQAPGKGLEWVSGIRPSGGTTVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARALPRSGYLISPHYYYYALDVWGQGT TVTVSS vLQSELTQPPSASGTPGQRVTISCSGSSSNIGRNYVYWYQQVPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLVISGLRSE DEADYYCAAWDASLRGVFGGGTKLTVLGHCDR 1 KYIMQ (Kabat 31-35) HCDR 2 GIRPSGGTTVYADSVKG (Kabat 50-65) HCDR 3ALPRSGYLISPHYYYYALDV (Kabat 95-102) LCDR 1 SGSSSNIGRNYVY (Kabat 24-34)LCDR 2 SNNQRPS (Kabat 50-56) LCDR 3 AAWDASLRGV (Kabat 89-97)

5) Analysis of Clone 3F7: FXIIa Inhibition by 3F7 mAb

To assess whether the 3F7 mAb inhibits FXIIa amidolytic activity in anin vitro assay, 3F7 Fab-phage was reformatted into full length humanIgG4/lambda antibody and purified using protocols described in Example3. Briefly, 1 μg of FXIIa was incubated in Nunc immunoplates in presenceor absence of rHA-Infestin-4, 3F7 mAb or control mAb (anti-human GCSFRantibody C1.2) in a volume of 160 μL for 5 min at 37° C. Forty μL ofsubstrate (4 mM S-2302) were added, and the plate was further incubatedat 37° C. for 15 min. The reaction was stopped by the addition of 40 μLof 20% acetic acid, and colour change was detected at 405 nm in platereader. The data was plotted using KaleidaGraph software with Sigmoidalcurve fit, and EC₅₀ value was recorded. As shown in FIG. 2, the 3F7antibody was found to effectively inhibit FXIIa amidolytic activity.

Example 2 Affinity Maturation of the 3F7 Antibody

The aim of the affinity maturation of 3F7 was to identify andcharacterise 3F7 mAb variants able to bind to human FXIIa with higheraffinity than the parental antibody.

Higher affinity variants have the potential to show improved inhibitionof FXIIa amidolytic activity. The method for the generation of Fab-phageaffinity maturation libraries (see Library construction below) isdependent on degenerate oligonucleotides annealing to a ssDNA templatewhich is then extended to make a double stranded form fortransformation. The size of the library is dependent upon transformationefficiency, and degeneracy of the primers used. The primers used (seebelow) covered a 19 amino acids combination (without cysteine).Libraries targeting 6 amino acid residues at a time were designed. Thetheoretical diversity of using trimer oligonucleotides for 6 residues is19⁶=4.7×10⁷.

1) Design of Affinity Maturation Libraries

For each phagemid, a germline stop template was created by replacing 18codons

(6 amino acid residues) in all CDRs, except CDR-L2, with TAA stopcodons. The linear design for the constructs is as follows:NcoI-VL-CL-linker-VH-SalI. Flanking NcoI and SalI sites were includedfor cloning into phage display pTac vector, containing remainingelements for phage display. The stop template versions named 3F7 H1, 3F7H2, 3F7 H3.1 and 3F7 H3.2 (heavy chain variable region) and 3F7 L1, 3F7L3.1, 3F7 L3.2 (light chain variable region) were produced by GeneArtand are shown in FIGS. 3 and 4 respectively.

2) Library Construction

Libraries were constructed using methods described by Sidhu et al.(Phage display for selection of novel binding peptides. Methods inEnzymology, 2000, vol. 238, p. 333-336) with “stop template” versions ofpTac-3F7 Fab. Each stop template was used as template for the Kunkelmutagenesis method (Kunkel et al., Rapid and efficient site-specificmutagenesis without phenotypic selection. Methods in Enzymology, 1987,vol. 154, p. 367-382) with mutagenic oligonucleoteides (Table 4)designed to simultaneously repair the stop codons and introducemutations at the designed sites. The mutagenesis reactions wereintroduced into E. coli SS320 by electroporation, and phage productionwas initiated with addition of M13-KO7 helper phage. After overnightgrowth at 30° C., the phage were harvested by precipitation withPEG/NaCl. The mutagenesis efficiencies were assessed by sequencing of 12clones randomly picked from each library, and ranged from 50 to 100%.Each library contained 0.75-3.75×10⁹ individual clones. Primer 3254(Table 2) was used to sequence clones from libraries L1, L3.1 and L3.2and primer Seq CL lambda (Table 2) was used to sequence clones fromlibraries H1, H2, H3.1 and H3.2.

TABLE 4  3F7 mutagenic trimer oligonucleotides used foraffinity maturation, where each “Nnn” designatesa triplet encoding one of 19 amino acids withoutcysteine (produced and supplied by Ella Biotech, Germany). 3F7 L1 5′GCTGTAGCGGTAGCAGCNnnNnnNnnNnnNnnNn nTATGTGTATTGGTATCAGCA  3′(SEQ ID NO: 24) 3F7 L3.1 5′ GATGAAGCCGATTATTATTGTNnnNnnNnnNnnNnnNnnCTGCGTGGTGTTTTTGGT 3′ (SEQ ID NO: 26) 3F7 L3.2 5′TTATTGTGCAGCATGGGATNnnNnnNnnNnnNnn NnnTTTGGTGGTGGCACCAAA 3′(SEQ ID NO: 28) 3F7 H1 5′ AGCAAGCGGTTTTACCTTTNnnNnnNnnNnnNnnNnnTGGGTTCGCCAGGCAC 3′ (SEQ ID NO: 16) 3F7 H2 5′GGAATGGGTTAGCGGTATTNnnNnnNnnNnnNnn NnnACCGTTTATGCAGATAGCG 3′(SEQ ID NO: 18) 3F7 H3.1 5′ TTATTATTGCGCACGTGCANnnNnnNnnNnnNnnNnnCTGATTTCTCCGCATTATTA 3′ (SEQ ID NO: 20) 3F7 H3.2 5′CACTGCCTCGTAGCGGTNnnNnnNnnNnnNnnNn nTATTATTATTATGCCCTGGAT 3′(SEQ ID NO: 22)

3) Library Panning

Libraries were cycled through five rounds of selection with decreasingconcentration of biotinylated FXIIa beta. The target concentration wasreduced 10-fold with each round, from 40 nM in Round 1 to 4 μM in Round5. Panning was carried out in solution with the biotinylated FXIIa beta.Phage samples were incubated with antigen diluted in 4% milk in PBST (or4% milk/PBST alone to make blank samples with no target) with rotationat RT for 1 hr. Dynal M-280 Streptavidin magnetic beads were blocked in5% skim milk/PBS for 30 min at 37° C. with horizontal shaking. Beadswere collected using MPS and phage/antigen mixture was added for 30minutes. Beads were then washed 10 times in PBST (KingFisher Long Wash),followed by a manual wash in PBS. Beads were finally resuspended in 500μL 50 mM DTT and incubated at 37° C. for 30 min with horizontal shaking.The eluted phage were collected, and added to 170 μL of neutralisationbuffer (0.351 g L-cysteine+5 mg BSA made up to 5 mL with 1 M Tris pH 8).330 μL of the eluted phage were used as input for the next round.Enrichment for each round of selection was calculated as ratio of elutedphage selected on target versus blank samples.

4) Analysis of Clones from 3F7 Affinity Maturation

At the completion of panning, a number of phage clones were selectedfrom each enriched library and sequenced using the primers detailedabove (library construction). Unique clones from each library were thenselected based on sequence and reformatted into fully human IgG4/lambdaantibodies for binding analysis. Affinity matured variants wereinitially screened using Biacore as unpurified cell culture supernatantto estimate binding affinities in comparison to parental 3F7 (asdescribed in Example 4(1)). Table 5 lists the antibodies that were foundto have a higher binding affinity to FXIIa beta than 3F7. The highestaffinity clones tended to come from the heavy chain CDR2 and the lightchain CDR 1 regions.

TABLE 5  Estimated binding affinities of 3F7 and affinity maturedvariants based on binding kinetics at a single FXIIa betaconcentration. All antibodies were tested as unpurifed IgG4molecules in cell culture supernatants on a Biacore A100instrument. Only variants with better affinity to FXIIa than  3F7 are shown. Refer to FIGS. 3 and 4 for library locations. mAb LibrarySequence SEQ ID NO KD (M) VR119 H2 NVPLYG 29 (residues 3-8) 1.31E−09VR112 H2 NVPVQG 30 (residues 3-8) 1.52E−09 VR115 H2 DIPTKG31 (residues 3-8) 1.56E−09 VR24 L1 EMTVHH 44 (residues 5-10) 1.63E−09VR110 H2 DMPTKG 32 (residues 3-8) 2.04E−09 VR107 H2 NPATRT33 (residues 3-8) 2.45E−09 VR06 L1 FSHPHH 45 (residues 5-10) 2.56E−09VR31 L3.1 ASWYND 52 (residues 1-6) 2.71E−09 VR108 H2 NPATKT34 (residues 3-8) 2.81E−09 VR103 H2 DVPVRG 35 (residues 3-8) 2.87E−09VR101 H2 NPATRS 36 (residues 3-8) 3.33E−09 VR16 L1 EFVEYN46 (residues 5-10) 3.63E−09 VR29 L3.1 ASWEIP 53 (residues 1-6) 3.89E−09VR05 L1 DTNSHH 47 (residues 5-10) 4.36E−09 VR12 L1 WTEQHN48 (residues 5-10) 4.45E−09 VR27 L3.1 ASWTNE 54 (residues 1-6) 4.64E−09VR10 L1 VMVTNH 49 residues 5-10) 4.97E−09 VR149 H3.2 YLMKKN39 (residues 7-12) 5.12E−09 VR58 L3.2 PQVRLA 59 (residues 5-10) 5.33E−09VR39 L3.1 ASWWND 55 (residues 1-6) 5.63E−09 VR167 H3.2 YLMKTG40 (residues 7-12) 5.80E−09 VR62 L3.2 QQVRLD 60 (residues 5-10) 5.81E−09VR109 H2 NPATNT 37 (residues 3-8) 5.98E−09 VR14 L1 GMVEQN50 (residues 5-10) 6.22E−09 VR46 L3.1 ASWELP 56 (residues 1-6) 6.67E−09VR148 H3.2 YLVKKQ 41 (residues 7-12) 6.93E−09 VR159 H3.2 YLVKHG42 (residues 7-12) 6.93E−09 VR53 L3.2 QQVRKT 61 (residues 5-10) 7.05E−09VR52 L3.2 ERVRLM 62 (residues 5-10) 7.10E−09 VR160 H3.2 YLMKPG43 (residues 7-12) 7.13E−09 VR17 L1 FKVEET 51 (residues 5-10) 7.15E−09VR63 L3.2 NQVRLG 63 (residues 5-10) 8.30E−09 VR41 L3.1 ASWSIP57 (residues 1-6) 9.14E−09 VR99 H2 NPATMT 38 (residues 3-8) 9.19E−09VR38 L3.1 ASWEVP 58 (residues 1-6) 9.23E−09 3F7 3.30E−08

Based on the results from the estimated binding affinity screening thebest 5 mabs were then purified and subjected to detailed bindingaffinity analysis (as described in example 4(2)). As shown in Table 6,these clones showed a 24 to 57-fold improvement in binding affinitycompared to parental 3F7.

TABLE 6 Detailed Biacore analysis of the binding affinity of purified3F7 and the top 5 affinity matured variants to FXIIa beta from Table 5.All antibodies were tested as fully human IgG4 molecules. Fold affinity3F7 Variant ka (1/Ms) kd (1/s) KD (M) improvement 3F7 1.2 × 10⁵ 1.1 ×10⁻³ 8.6 × 10⁻⁹ 1 VR115 1.7 × 10⁵ 2.5 × 10⁻⁵ 1.5 × 10⁻¹⁰ 57 VR112 2.5 ×10⁵ 4.3 × 10⁻⁵ 1.7 × 10⁻¹⁰ 51 VR24 2.4 × 10⁵ 6.4 × 10⁻⁵ 2.6 × 10⁻¹⁰ 33VR110 1.1 × 10⁵ 3.9 × 10⁻⁵ 3.5 × 10⁻¹⁰ 25 VR119 1.6 × 10⁵ 5.9 × 10⁻⁵ 3.6× 10⁻¹⁰ 24

Example 3 IgG Production and Purification of Phage-Derived Antibodies ofthe Invention 1) Mammalian Expression Vector Construction

The mammalian expression vectors were constructed using standardmolecular biology techniques by cloning the entire light chain (variableand constant domains) and the variable domain of the heavy chain fromthe selected phage-derived Fab constructs into the pRhG4 vector aspreviously described (Jostock et al 2004. Rapid generation of functionalhuman IgG antibodies derived from Fab-on-phage display libraries. JImmunol Methods, 289; 65-80).

2) Cell Culture

Serum-free suspension adapted 293-T cells were obtained from GenechoiceInc. Cells were cultured in FreeStyle™ Expression Medium (Invitrogen)supplemented with penicillin/streptomycin/fungizone reagent(Invitrogen). Prior to transfection the cells were maintained at 37° C.in humidified incubators with an atmosphere of 8% CO₂.

3) Transient Transfection

The transient transfection of the mammalian expression vectors using293-T cells was performed using 293fectin transfection reagent(Invitrogen) according to the manufacturer's instructions. The light andheavy chain expression vectors were combined and co-transfected with the293-T cells. Cells (1000 ml) were transfected at a final concentrationof 1×10⁶ viable cells/ml and incubated in a Cellbag 2 L (Wave Biotech/GEHealthcare) for 5 days at 37° C. with an atmosphere of 8% CO₂ on a 2/10Wave Bioreactor system 2/10 or 20/50 (Wave Biotech/GE Healthcare). Theculture conditions were 35 rocks per minute with an angle of 8°.Pluronic® F-68 (Invitrogen), to a final concentration of 0.1% v/v, wasadded 4 hours post-transfection. 24 hours post-transfection the cellcultures were supplemented with Tryptone N1 (Organotechnie, France) to afinal concentration of 0.5% v/v. The cell culture supernatants wereharvested by centrifugation at 2500 rpm and were then passed through a0.45 μM filter (Nalgene) prior to purification.

4) Analysis of Protein Expression

After 5 days 20 μl of culture supernatant was electrophoresed on a 4-20%Tris-Glycine SDS polyacrylamide gel and the antibody was visualised bystaining with Coomassie Blue reagent.

5) Antibody Purification

Monoclonal antibodies were purified using tandem protein A affinitychromatography and desalting column chromatography. Chromatography using

Hitrap MabSelect sure (1 ml, GE Healthcare, UK) and Desalting (HiPrep26/10, GE Healthcare, UK) resins were developed using an AKTA express(GE Healthcare, UK) as per manufacturers recommended method. Briefly,equilibration of the Protein A affinity column was performed in 1×MT-PBSbuffer. The filtered conditioned cell culture media (500 ml) was appliedto the column at 1 ml/min and washed sequentially with 1×MT-PBS (10 ml)and 10 mM Tris, 0.5M Arginine, 150 mM NaCl pH 7.2 (80 ml). The boundantibody was then eluted with 0.1M Na Acetate pH 3.0 (8 ml) andimmediately applied to the desalting column. The antibody concentrationwas determined chromatographically by comparison to control antibodystandards. Protein fractions were pooled and concentrated using anAmicon UltraCel 50K centrifugal device (Millipore) prior to sterilefiltration using 0.22 um filters.

The purity of the antibody was analysed by SDS-PAGE, where 2 μg proteinin reducing Sample Buffer (Invitrogen, CA) was loaded onto a NovexNuPAGE 4-12% Bis-Tris Gel (Invitrogen, CA) and a constant voltage of200V was applied for 40 minutes in an XCell SureLock Mini-Cell(Invitrogen, CA) with NuPAGE MES SDS running buffer before beingvisualised using Coomassie Stain, as per the manufacturer'sinstructions.

Example 4 Antibody Affinity Determination—Biacore Analysis

1) Estimated Binding Affinities from Unpurifed Antibody Supernatants

Anti-human (Goat anti-human IgG (gamma) mouse adsorbed, Invitrogen, CatNo. H10500) was chemically immobilised on a CM-5 sensor surface usingamine coupling chemistry. Culture supernatants were diluted 1/60 withrunning buffer before capture. Antibodies were captured for 180 secondsrepresenting an average capture of 800 response units (RU). FXIIa betawas then injected at zero and 100 nM for 180 seconds, and dissociatedfor 180 seconds. All assays were conducted on a Biacore A100 instrumentat 37 degrees Celsius and the data fitted to a 1:1 kinetic model.

2) Detailed Binding Affinity Analysis

Anti-human (Goat ant-Human IgG (gamma) mouse adsorbed, Invitrogen, CatNo. H10500) or anti mouse Fc specific antibody (Jackson Immuno ResearchLabs inc. Cat No. 515-005-071) was chemically immobilised on a CM-5sensor surface using amine coupling chemistry. The immobilisedantibodies were then used to capture anti-FXII/FXIIa mAbs from solution.

Human FXII or FXIIa beta was then injected over captured antibody atvarious concentrations for detailed binding kinetics. Responses from areference flow cell (in which mAb was not captured, but otherwisetreated identically), were subtracted. The responses from a blankinjection were then subtracted from the resultant sensorgrams.

The final corrected responses were fitted using non-linear regression toa model describing 1:1 kinetics, including a term for mass transportlimitation. The Rmax value was fitted locally, to account for slightdeviations in the level of mAb captured. Association rate (ka),dissociation rate (kd) and equilibrium dissociation constant (KD) weredetermined.

For detailed binding kinetics FXII was injected at 0, 15.1, 31.25, 62.5,125, 250, and 500 nM, in duplicate and FXIIa beta was injected at 0,1.25, 2.5, 5, 10, 20 and 40 nM, with 10 nM in duplicate.

For the 3F7 antibody, regeneration was performed after each cycle with a90 second injection of 100 mM H₃PO₄. For mab OT-2, regeneration wasperformed after each cycle with a 60 second injection of 25 mM glycine,pH 1.7, followed by a 30 second injection of 25 mM glycine, pH 8.6. Allassays were conducted at 25° C.

Example 5 Comparison of 3F7 with Other Antibody Inhibitors of FXIIaAmidolytic Activity

A review of the relevant scientific literature revealed that although anumber of antibodies have been described which can modulate FXIIactivity, the majority of these are either directed to the heavy chainand prevent the initial contact activation of FXII or are directed tothe light chain and appear to only partially inhibit FXII amidolyticactivity. The aim of this work was to compare 3F7 to antibody OT-2 whichhas been claimed to completely block the amidolytic activity of FXIIa(Dors et al., A novel sensitive assay for functional FXII based on thegeneration of kallikrein-C1-inhibitor complexes in FXII deficient plasmaby glass-bound Factor XII. Thrombosis and Haemostasis, 1992, vol. 67, p.644-648; Citarella et al., Structure/function analysis of human factorXII using recombinant deletion mutants. European Journal ofBiochemistry, 1996, vol. 238, p. 240-249).

1) Inhibition of FXIIa Amidolytic Activity with 3F7 and OT-2 Antibodies

The activity of 3F7 and OT-2 antibodies was compared in an in vitroFXIIa amidolytic activity assay, essentially as described in Example1(5). Both antibodies were able to completely block the amidolyticactivity of FXIIa (FIG. 5).

2) Biacore Analysis of 3F7 and OT-2 mAbs Binding to FXII and ActivatedFXIIa Beta

Whilst both 3F7 and OT-2 were shown to completely block the amidolyticactivity of FXIIa, 3F7 showed a small but reproducible ˜2-fold higherpotency in this assay. To determine if 3F7 and OT-2 share a similarepitope on FXIIa we initially performed a competition ELISA with theseantibodies and showed they were able to effectively compete with eachother for binding to FXIIa (data not shown). To further characterize thecomparative binding of these antibodies to FXII we performed Biacoreexperiments with both antibodies against unactivated FXII andcatalytically active FXIIa beta. The results of this experiment areshown in Table 7 and demonstrate that whilst OT-2 shows equivalentbinding affinity to FXII or activated FXIIa beta, 3F7 shows a clearpreference for binding to the activated form of FXII (FXIIa). Theseresults show that whilst both antibodies appear to bind to similarregions on the light chain of FXII they do not appear to share anidentical epitope. The ability of 3F7 to preferentially bind toactivated FXII may confer a pharmokinetic and/or pharmacodynamicadvantage.

TABLE 7 Detailed Biacore analysis of the binding affinity of thepurified IgG monoclonal antibodies 3F7 and OT-2 to FXIIa beta. mAb FXIIKD (nM) FXIIαβ KD (nM) 3F7  121 ± 19 (N = 3)  6.2 ± 0.2 (N = 3) OT-20.69 ± 0.25 (N = 3) 0.76 ± 0.077 (N = 3)

Example 6 Identifying Key FXIIa Residues Involved in the Binding of 3F7

Having screened 3F7 for its ability to inhibit the activity of FXIIafrom a number of species (data not shown), we determined 3F7 to behighly potent against mouse and human FXIIa, but not rat FXIIa. Usingthis information we investigated which key residues within the FXIIalight chain may be involved in the 3F7 epitope by generating arecombinant murine FXII (which is recognised by 3F7 using Westernanalysis) and mutating various residues that differed from the rat aminoacid (see FIG. 6). As the result shows, mutating either position 398 or438 abolishes the binding of 3F7.

1. Construction and Expression of Wild-Type and Mutant Murine Factor XII(Mu-FXII)

A cDNA encoding the entire Mu-FXII protein (GenBank Accession no.NM_(—)021489) was obtained from GeneART AG (Regensberg, Germany). ThiscDNA was used as a template to make the following separate residuechanges by standard PCR techniques: a) N376D, b) A385D, c) N398K, d)W420R, e) R427H, f) I438A, g) Q450R, h) delE451, i) S452G, j) K453R,T454K, k) G472S, l) N516S, m) T538A and n) A589D. With the exception off) I438A, these residue changes corresponded to a switch from the mouseresidue to its rat orthologue (GenBank Accession no. NM_(—)001014006)(see FIG. 6A). In the case of h), this involved a deletion of Glu451.One further mutant (o) was generated, a multiple mutant involving themurine to rat amino acid changes E552D, T555V and A556T. All constructswere modified at the 3′ end to encode a C-terminal 8×His-tag, clonedinto the mammalian expression vector pcDNA3.1 (Invitrogen, Carlsbad,USA) and the sequence validated by DNA sequence analysis.

Freestyle™ 293 suspension cells (Invitrogen) were grown to 1.1×10⁶cells/ml in 5 ml Freestyle Expression media (Invitrogen). 7 μL 293Fectin(Invitrogen) transfection reagent was pre-incubated for 5 minutes with167 μL Opti-MEM I medium (Invitrogen), then added to 5 μg plasmid DNAencoding wild-type or mutant Mu-FXII and the mixture incubated for afurther 20 minutes. The DNA-293Fectin complex was added to the cellswhich were cultured for 6 days at 37° C., 8% CO₂ in a shaking incubatorat 250 rpm. Culture supernatants were harvested by centrifugation at2000 rpm for 5 minutes and stored at 4° C. for analysis.

2. Western Blotting

Supernatants containing recombinant wild-type or mutant mu-FXII wereadded to equal volumes of 2× non-reducing sample buffer, incubated at80° C. for 10 minutes and then loaded onto pre-cast 4-12% Bis-Tris gels(Invitrogen) and electrophoresed for 1 hour at 200V. Proteins were thentransferred electrophoretically onto nitrocellulose filters and blockedfor 1 hour in 5% Milk powder in Tris-buffered saline with 0.05% Tween-20(TTBS). Filters were then incubated for 1 hour with either 3F7 mAb or ananti-His mAb 3H3 (both at 1 mg/mL in TTBS with 5% Milk powder), washedthoroughly with TTBS, then incubated for a further hour with anti-humanIgG-FITC or anti-mouse IgG-FITC, respectively (Millipore, USA; both at0.25 mg/ml in TTBS with 5% Milk powder). Following further washing ofmembranes in TTBS, Ab-FITC bound proteins were visualized using aTyphoon variable mode analyzer (GE Healthcare, USA). The results areshown in FIG. 6B. The binding of 3F7 is abolished when residues 398 and438 of the mouse sequence are mutated, indicating that these tworesidues may be part of the epitope of mAb 3F7.

Example 7 Prevention of FeCl₃-Induced Arterial Thrombosis in Mice withby Intravenous Treatment with Monoclonal Antibody 3F7

Previous studies (e.g. disclosed in WO2006066878) have shown thatinhibition of FXIIa prevented FeCl₃-induced arterial thrombosis in mice.The goal of this study was to explore whether mice are also protectedagainst arterial thrombosis by treatment with a specific monoclonalantibody directed against coagulation factor XIIa (MAb 3F7).

Methods

Treatment groups were as shown in Table 8:

TABLE 8 Treatment groups No. Treatment Dose/volume/schedule/route N (f)1 Isotonic N.a.¹/0.1 mL/20 g b.w./t = −15 min./i.v. 25 saline 2 MAb 3F730 mg/kg/0.1 mL/20 g b.w./t = −15 min./i.v. 10 3 MAb 3F7 20 mg/kg/0.1mL/20 g b.w./t = −15 min./i.v. 5 4 MAb 3F7 10 mg/kg/0.1 mL/20 g b.w./t =−15 min./i.v. 10 5 MAb 3F7 5 mg/kg/0.1 mL/20 g b.w./t = −15 min./i.v. 106 MAb 3F7 2.5 mg/kg/0.1 mL/20 g b.w./t = −15 min./i.v. 10 7 MAb 3F7 1mg/kg/0.1 mL/20 g b.w./t = −15 min./i.v. 10 8 MAb 3F7 0.5 mg/kg/0.1mL/20 g b.w./t = −15 min./i.v. 10 9 Control 30 mg/kg/0.2 mL/20 g b.w./t= −15 min./i.v. 10 MAb ¹N.a. = not applicable

Mice of strain NMRI, obtained from Charles River Laboratories, female,aged 6-8 weeks, weighing between 25 and 39 g, received a single i.v.injection of the treatment solution as listed in Table 8 at t=−15 min indeep anesthesia. Thereafter, the effects of the treatment on thethrombotic occlusion rate were quantified. Baseline blood flow wasdetermined by placing an ultrasonic flow probe around the exposedarteria carotis. To initiate thrombosis, a 0.5 mm² (0.5×1.0 mm) patch offilter paper saturated with 10% ferric chloride solution was placed onthe arteria carotis downstream from the flow probe at t=0 min. After 3minutes the filter paper was removed and blood flow was monitored for 60minutes to determine the occurrence of thrombotic occlusions.

Following the 60 minutes observation period, blood samples were takenfrom study animals (anticoagulant: 10% citrate). Thereafter, plasma wasprepared according to standard methods, and deep frozen (−80° C.±10° C.)until determination of aPTT (activated partial thromboplastin time), PT(prothrombin time) and FXIIa-activity.

Determination of the aPTT:

The aPTT was determined by adding 50 μL of study plasma samples (seeabove) to 50 μL Pathromtin SL (Siemens HealthCare Diagnostics ProductsGmbH, Marburg, Germany) followed by an incubation phase of 120 secondsat 37° C. Subsequently, 50 μL of a calcium chloride solution (25 mM,Siemens HealthCare Diagnostics Products GmbH, Marburg, Germany) wasadded to start the reaction.

Determination of the PT:

The PT was determined by adding 50 μL of study plasma samples (seeabove) to 100 μL of the activation reagent Thromborel S (SiemensHealthCare Diagnostics Products GmbH, Marburg, Germany) after 15 secondsincubation time at 37° C.

Determination of FXIIa-Activity:

The FXIIa-activity was determined by using an aPTT-based assay andcompared to a reference curve obtained with dilutions of standard humanplasma and FXII-deficient plasma (Siemens HealthCare DiagnosticsProducts GmbH, Marburg, Germany). 50 μL of the study plasma samples (seeabove), which were pre-diluted 1:5 with imidazol buffer solution(Siemens HealthCare Diagnostics Products GmbH, Marburg, Germany), wereadded to 50 μL of FXII-deficient plasma. After an incubation time of 30seconds at 37° C., 50 μL Pathromtin SL (Siemens HealthCare DiagnosticsProducts GmbH, Marburg, Germany) was added and the solution thereafterincubated for 120 seconds at 37° C. Subsequently, 50 μL of a calciumchloride solution (25 mM, Siemens HealthCare Diagnostics Products GmbH,Marburg, Germany) was added to start the reaction.

All three analyses were performed in a BCT (Behring Coagulation Timer;Siemens HealthCare Diagnostics Products GmbH, Marburg, Germany) in linewith the conditions suggested by the supplier of respective assayreagents (Siemens HealthCare Diagnostics Products GmbH, Marburg,Germany).

Results:

Intravenous injection of 30 mg/kg, 20 mg/kg, 10 mg/kg and 5 mg/kg MAb3F7 resulted in a complete protection from FeCl₃-induced occlusion ofthe arteria carotis of mice (Table 9, FIG. 7). At decreasing doses (i.e.2.5-0.5 mg/kg), occlusion rates increased while times to occlusiondecreased dose-dependently (Table 9, FIG. 7). Compared to controls, PTwas unchanged (Table 10, FIG. 9) while aPTT was prolonged about fourfoldat the high doses (Table 10, FIG. 8). FXIIa-activity was nearlycompletely inhibited at a dose of 10 mg/kg and above, and still halvedat a dose of 0.5 mg/kg (Table 10, FIG. 10). Furthermore, aPTT decreasedwhile FXIIa-activity increased dose-dependently at decreasing doses ofthe MAb 3F7 (Table 10, FIGS. 8 and 10). The control MAb showed noprotection from FeCl₃-induced occlusion of the arteria carotis and aPTT,PT and FXIIa-activity values were unchanged (Tables 9, 10, FIGS. 7 to10).

TABLE 9 Occlusion rates No. Treatment Occlusion rate 1 Isotonic saline21/25 (84%)  2 MAb 3F7 0/10 (0%)  30 mg/kg 3 MAb 3F7 0/5 (0%) 20 mg/kg 4MAb 3F7 0/10 (0%)  10 mg/kg 5 MAb 3F7 0/10 (0%)  5 mg/kg 6 MAb 3F7 3/10(30%) 2.5 mg/kg 7 MAb 3F7 4/10 (40%) 1 mg/kg 8 MAb 3F7 6/10 (60%) 0.5mg/kg 9 Control MAb 8/10 (80%) 30 mg/kg

TABLE 10 PT, aPTT and FXIIa-activity (mean ± SD) FXIIa- No. Treatment PTaPTT activity 1 Isotonic saline 8.99 ± 1.13 31.19 ± 3.97 71.13 ± 14.64 2MAb 3F7 10.58 ± 1.12  116.70 ± 29.96 0.63 ± 1.16 30 mg/kg 3 MAb 3F711.68 ± 0.98  137.90 ± 7.74  0.00 ± 0.00 20 mg/kg 4 MAb 3F7 9.74 ± 0.57124.70 ± 24.41 0.94 ± 1.29 10 mg/kg 5 MAb 3F7 10.43 ± 0.92   91.72 ±16.89 3.17 ± 0.92 5 mg/kg 6 MAb 3F7 9.14 ± 0.33  69.02 ± 11.05 7.68 ±1.59 2.5 mg/kg 7 MAb 3F7 9.51 ± 0.61 39.84 ± 5.83 30.02 ± 10.00 1 mg/kg8 MAb 3F7 9.61 ± 0.60 35.89 ± 3.73 37.22 ± 7.92  0.5 mg/kg 9 Control MAb7.56 ± 0.28 29.33 ± 2.52 59.70 ± 12.54 30 mg/kg

Discussion:

This study demonstrated that mice were fully protected against arterialthrombosis after intravenous treatment with the MAb 3F7 at a dose of 5mg/kg or higher. At decreasing doses, occlusion rates increased whiletimes to occlusion decreased dose-dependently. Compared to controls, PTwas unchanged while aPTT and FXIIa-activity were dose-dependentlyprolonged and decreased, respectively. In summary, MAb 3F7 demonstrateda remarkable efficacy profile and a desirable dose-responserelationship.

Example 8 Effects of Anti-FXIIa Monoclonal Antibody 3F7 on Hemostasis ina Subaquatic Bleeding Model in Mice

Example 7 had demonstrated that MAb 3F7 fully prevents FeCl₃-inducedarterial thrombosis in mice at doses of 30-5 mg/kg. In addition to thiseffect, FXIIa-activity was nearly completely inhibited and aPTTprolonged up to fourfold at these protective doses. In order to clarifythe question whether such effects may influence physiologicalhemostasis, the aim of this study is to investigate MAb 3F7 with regardto its effect on hemostasis in the murine tail tip bleeding model at thelowest fully protective dose (i.e. 5 mg/kg) as well as 5 fold beyondthis dose (i.e. 25 mg/kg).

Methods

TABLE 11 Treatment groups No. Treatment Dose/volume/schedule/route N(m/f) 1 Isotonic N.a¹./0.1 mL/20 g b.w./t = −5 min./i.v. 10 (0/10)saline 2 MAb 3F7 5 mg/kg/0.1 mL/20 g b.w./t = −5 min./i.v. 10 (0/10) 3MAb 3F7 25 mg/kg/0.1 mL/20 g b.w./t = −5 min./ 10 (0/10) i.v. ¹N.a = notapplicable

Female NMRI mice were obtained from Charles River Laboratories(Kisslegg). They were 6 to 8 weeks old and weighed 25 to 32 g.

Hemostasis was determined in a subaquatic model. In brief, tail tipbleeding parameters were determined by quantifying time to hemostasisand blood loss. The volume of total blood loss was calculated bymeasuring the hemoglobin present in the saline used for submersion ofthe tail tip. The hemoglobin of the animals was taken into considerationaccordingly. The tail tip cut was performed with a scalpel knife underdeep anesthesia (Narcoren), removing about 3 mm of the tail tip.Immediately upon lesion, the tail tip was submerged in saline, which waskept at the physiological body temperature of the mice using a waterbath. The observation period to monitor bleeding was 30 min. All testarticles were administered i.v. at 5 min. prior to the start of theobservation period (tail cut).

Results:

Independent of group, all animals showed hemostasis within theobservation period (Table 12). Time to hemostasis and total blood lossdid not differ between the groups (Tables 12 and 13, FIGS. 11 to 14;Kruskal-Wallis test: p>0.05).

TABLE 12 Frequency and Time to Hemostasis within 30 minutes followingtreatment with MAb 3F7 (n = 10/group) Time to hemostasis Mean ±Frequency of SD Min. Med. Max. Treatment hemostasis (sec.) (sec.) (sec.)(sec.) Isotonic saline 10/10 (100%) 157 ± 94  70 125 360 MAb 3F7  5mg/kg 10/10 (100%) 178 ± 185 60 98 660 MAb 3F7 25 mg/kg 10/10 (100%) 196± 144 30 163 450

TABLE 13 Total blood loss following treatment with MAb 3F7 (n =10/group) Median Max. Treatment Mean ± SD (μL) Min. (μL) (μL) (μL)Isotonic saline 12.3 ± 9.5  2.1 10.5 27.0 MAb 3F7  5 mg/kg 7.0 ± 7.1 0.64.2 23.3 MAb 3F7 25 mg/kg  9.9 ± 10.3 2.1 5.1 30.8

Discussion:

From the results of this study, it can be concluded that the two applieddoses of MAb 3F7 (5 and 25 mg/kg), potently preventing FeCl₃-inducedarterial thrombosis in mice, had no effects on physiological hemostasisusing the murine tail tip bleeding model.

Example 9 Comparison of aPTT of 3F7 and Affinity-Matured Versions

The activated partial thromboplastin time (aPTT) was determined instandard human plasma (SHP, Dade Behring), where different amounts ofthe respective inhibitor were added into physiological saline to a totalvolume of 200 μL. 50 μL of this solution were added to 50 μL PathromtinSL (Dade Behring) and incubated for 120 sec at 37° C. Subsequently, 50μL of a calcium chloride solution (25 mM) were added to start thereaction.

The procedure was performed in a BCS XP (Behring Coagulation System)according to the conditions suggested by the manufacturer.

The aPTT of OT-2, MAb 3F7 and affinity-matured versions of MAb 3F7 wascompared. The results are shown in FIG. 15. The affinity-maturedversions of MAb 3F7 were significantly more active than OT-2 and theoriginal MAb 3F7.

Example 10 Comparison of the Inhibition of Factor XIIa-Alpha byDifferent Antibodies

An inhibition assay was performed, essentially as described in Example1(5) above. In this case, 3F7, the affinity-matured 3F7 derivatives andOT-2 were compared in different molar ratios to human Factor XIIa-alpha,ranging from 1:0.1 to 1:10. The data are shown in Table 14 below and inFIG. 16. 3F7 and the affinity-matured derivatives showed betterinhibition than OT-2., and a higher amount of OT-2 was required toachieve maximal inhibition than of 3F7 and derivatives thereof.

TABLE 14 Antibody Ratio FXIIa-alpha:Antibody % Inhibition 3F7   1:0.135.5   1:0.2 62.5   1:0.5 91.9 1:1 97.9 1:2 100 1:5 100  1:10 100 3F7  1:0.1 38.3   1:0.2 66.8   1:0.5 91.4 1:1 96.1 1:2 100 1:5 100  1:10100 VR115 0.1:1   39.4 0.2:1   72.8 0.5:1   100 1:1 100 1:2 100 1:5 100 1:10 100 VR112 0.1:1   39.9 0.2:1   68.7 0.5:1   99.7 1:1 100 2:1 1005:1 100 10:1  100 VR110 0.1:1   33.7 0.2:1   66.9 0.5:1   100 1:1 1002:1 100 5:1 100 10:1  100 VR24 0.1:1   34.5 0.2:1   67.3 0.5:1   99.71:1 100 2:1 100 5:1 100 10:1  100 OT-2 0.1:1   21.6 0.2:1   37.7 0.5:1  76.6 1:1 92.7 2:1 96.9 5:1 100 10:1  100 Infestin control 1:1 90.0

1. An anti-Factor XII/XIIa monoclonal antibody or antigen-bindingfragment thereof that has a more than 2 fold higher binding affinity tohuman Factor XIIa-beta than to human Factor XII and that is capable ofinhibiting the amidolytic activity of human Factor XIIa at aconcentration of 100 nM or lower in an in vitro amidolytic activityassay by 80% or more.
 2. An anti-Factor XII/XIIa monoclonal antibody orantigen-binding fragment thereof that inhibits the amidolytic activityof Factor XIIa-alpha by more than 50% in an in vitro amidolytic activityassay when used at a molar ratio of FXIIa-alpha to antibody of 1:0.2. 3.The antibody or antigen-binding fragment claim 1, wherein the antibodyor antigen-binding fragment binds murine FXII/FXIIa; and wherein theantibody or antigen-binding fragment binds to a polypeptide comprisingSEQ ID NO: 2 in which (a) the asparagine residue at position 398 of SEQID NO: 2 is substituted for lysine; or (b) the isoleucine residue atposition 438 of SEQ ID NO: 2 is substituted for alanine, and wherein theaffinity of the antibody or antigen-binding fragment for saidpolypeptide is lower than the affinity of the antibody orantigen-binding fragment for a polypeptide comprising SEQ ID NO: 2without said substitution.
 4. The antibody or antigen-binding fragmentof claim 1, wherein the antibody or antigen-binding fragment comprises aheavy chain variable (vH) region comprising an amino acid sequence morethan 85% identical to the sequence of SEQ ID NO:
 4. 5. The antibody orantigen-binding fragment of claim 1, wherein the antibody orantigen-binding fragment comprises a light chain variable (vL) regioncomprising an amino acid sequence more than 85% identical to thesequence of SEQ ID NO:
 5. 6. The antibody or antigen-binding fragment ofclaim 1, wherein the antibody or antigen-binding fragment comprises aheavy chain CDR1 at least 80% identical to the sequence of SEQ ID NO: 6,a heavy chain CDR2 at least 60% identical with SEQ ID NO: 7, and a heavychain CDR3 at least 80% identical to the sequence of SEQ ID NO:
 9. 7.The antibody or antigen-binding fragment of claim 1, wherein theantibody comprises a light chain CDR1 at least 50% identical to SEQ IDNO: 11, a light chain CDR2 comprising SEQ ID NO: 12, and a light chainCDR3 comprising SEQ ID NO:
 14. 8. The antibody or antigen-bindingfragment of claim 1, wherein the antibody or antigen-binding fragmentbinds human Factor XIIa-beta with a K_(D) of better than 10⁻⁷ M.
 9. Theantibody or antigen-binding fragment of claim 1, wherein the antibody orantigen-binding fragment competes with Infestin for binding to humanFactor XIIa-beta.
 10. The antibody or antigen-binding fragment of claim1, wherein the antibody or antigen-binding fragment is a human IgG orvariant thereof.
 11. The antibody or antigen-binding fragment of claim10, wherein the IgG is IgG4.
 12. A nucleic acid encoding the antibody orantigen-binding fragment of claim
 1. 13. A vector comprising the nucleicacid of claim 12, operably linked to a promoter sequence.
 14. A cellcomprising the vector of claim
 13. 15. A method of producing theantibody or antigen-binding fragment of claim 1, comprising culturingthe cell of claim 14 to express the antibody or antigen-binding fragmentand purifying the antibody or antigen-binding fragment from the culturesupernatant.
 16. (canceled)
 17. A method of treating venous, arterial orcapillary thrombus formation, thrombus formation in the heart,thromboembolism, thrombus formation during and/or after contacting bloodwith artificial surfaces, intraluminal thrombi; interstitial lungdisease, inflammation, a neurological inflammatory disease, complementactivation, fibrinolysis, angiogenesis, or a disease related toFXII/FXIIa-induced kinin formation or FXII/FXIIa-mediated complementactivation in a subject in need thereof, the method comprisingadministering to the subject an effective amount of the antibody orantigen-binding fragment of claim
 1. 18. The method of claim 17, whereinthe subject suffers from venous or arterial thrombus formation selectedfrom stroke, myocardial infarction, deep vein thrombosis, portal veinthrombosis, thromboembolism, renal vein thrombosis, jugular veinthrombosis, cerebral venous sinus thrombosis, Budd-Chiari syndrome andPaget-Schroetter disease.
 19. The method of claim 17, wherein thesubject suffers from a disease related to FXII/FXIIa-induced kininformation selected from hereditary angioedema, bacterial infection ofthe lung, trypanosoma infection, hypotensive shock, pancreatitis, chagasdisease, articular gout, arthritis, disseminated intravascularcoagulation (DIC) and sepsis.
 20. The method of claim 17, wherein thesubject suffers from an interstitial lung disease selected fromfibroproliferative and idiopathic pulmonary fibrosis.
 21. The method ofclaim 17, wherein the subject suffers from thrombus formation duringand/or after contacting blood of the subject with one or more artificialsurfaces during and/or after a medical procedure performed on saidsubject, and wherein said antibody or antigen-binding fragment isadministered before and/or during and/or after said medical procedure,and further wherein (i) the artificial surface is exposed to at least80% of the blood volume of the subject and the artificial surface is atleast 0.2 m² or (ii) the artificial surface is a container forcollection of blood outside the body of the subject or (iii) theartificial surface is a stent, valve, intraluminal catheter, or a systemfor internal assisted pumping of blood.
 22. A medical device comprisingone or more surfaces coated with the antibody or antigen-bindingfragment of claim 1, wherein the device is a cardiopulmonary bypassmachine, an extracorporeal membrane oxygenation system for oxygenationof blood, a device for assisted pumping of blood, a blood dialysisdevice, a device for the extracorporeal filtration of blood, arepository for use in the collection of blood, an intraluminal catheter,a stent, an artificial heart valve.
 23. A method of treating ischemia ina subject in need thereof comprising administering an effective amountof the antibody or antigen-binding fragment of claim 1 to the subjectbefore, during, and/or after a medical procedure that could result inischemia in at least one target organ of the subject, wherein themedical procedure comprises contact with at least one of: (a) heart, (b)at least one blood vessel chosen from: the aorta, the aortic arch, acarotid artery, a coronary artery, brachiocephalic artery,vertebrobasilar circulation, an intracranial artery, a renal artery, ahepatic artery, a mesenteric artery, and/or a blood vessel of thearterial system cranial to the heart, and (c) at least one venous bloodvessel if the subject has a known septal defect; and wherein the medicalprocedure further comprises release of at least one embolus in at leastone blood vessel in the body that could result in ischemia in at leastone target organ of the subject.
 24. A method of treating increasedretinal vascular permeability resulting from progressive retinopathy,sight-threatening complication of retinopathy, macular edema,non-proliferative retinopathy, proliferative retinopathy, retinal edema,diabetic retinopathy, hypertensive retinopathy, and retinal trauma, in asubject in need thereof, the method comprising administering aneffective amount of the antibody or antigen-binding fragment of claim 1to the subject.
 25. A pharmaceutical composition comprising the antibodyor antigen-binding fragment of claim 1 and at least one pharmaceuticallyacceptable excipient.
 26. The device of claim 22, wherein one or more oftubing, cannulae, a centrifugal pump, a valve, a port, and/or a diverteris coated with the antibody or antigen-binding fragment.